CN114667343A - Escherichia coli composition and method thereof - Google Patents

Escherichia coli composition and method thereof Download PDF

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CN114667343A
CN114667343A CN202080076411.2A CN202080076411A CN114667343A CN 114667343 A CN114667343 A CN 114667343A CN 202080076411 A CN202080076411 A CN 202080076411A CN 114667343 A CN114667343 A CN 114667343A
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polypeptide
formula
coli
cell
fragment
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A·S·安德森
陈伟
L·O·乔罗
朱晓临
R·G·K·唐纳德
M·C·格里弗
顾建新
管泽强
金进焕
S·柯达里
S·E·隆伯克
J·A·洛特文
N·麦钱特
J·K·莫兰
R·潘
A·K·普拉萨德
M·E·鲁彭
S·辛格
D·R·斯特德
K·K·高根
车晔
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Pfizer Inc
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/385—Haptens or antigens, bound to carriers
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    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/245—Escherichia (G)
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    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

In one aspect, the invention relates to polypeptides derived from E.coli and fragments thereof, including compositions and methods thereof. Also disclosed herein are compositions comprising polypeptides derived from escherichia coli and fragments thereof; and modified O-polysaccharide molecules derived from Escherichia coli lipopolysaccharides and conjugates thereof. In yet another aspect, disclosed herein is a mammalian host cell comprising one or more sequences encoding polypeptides derived from e.

Description

Escherichia coli composition and method thereof
Cross Reference to Related Applications
This application claims benefit of U.S. provisional application No. 62/929,505 filed on 1/11/2019, U.S. provisional application No. 63/045,038 filed on 26/6/2020, and U.S. provisional application No. 63/081,629 filed on 22/9/2020. The entire contents of each of the foregoing applications are incorporated herein by reference.
Reference to sequence listing
This application is submitted electronically via the EFS website, including electronically submitted sequence listings in the txt format. The txt file contains a sequence listing entitled "PC 072517_03_ SEQ _ List _ st25. txt", created at 9/18/2020, and is 152KB in size. The sequence listing contained in the txt file is part of this specification and is incorporated herein by reference in its entirety.
Technical Field
The present invention relates to Escherichia coli compositions and methods thereof.
Background
The bacterial pilus adhesins FimH and FmlH allow e.coli to exploit different urinary tract microenvironments by recognizing specific host cell glycoproteins. FimH binds to the mannosylated urokinase protein (uroplakin) receptor in the urothelium, while FmlH binds to galactose or N-acetylgalactosamine O-glycans on epithelial surface proteins in the kidney and inflamed bladder. FimH pili also play a role in colonization of the gut by enterotoxigenic escherichia coli (ETEC) and multidrug resistant invasive escherichia coli by binding to highly mannosylated proteins on the intestinal epithelial cells.
Full-length FimH consists of two domains: an N-terminal lectin domain and a C-terminal pilin domain, said domains being linked by a short linker. The lectin domain of FimH comprises a carbohydrate recognition domain that is responsible for binding to mannosylated urolytic protein 1a on the surface of urothelial cells. The pilin domain is anchored to the core of the pilus by the donor chain of the subsequent FimG subunit, a process called donor chain complementation.
The conformational and ligand binding properties of FimH's lectin domain are controlled by the allosteric control of FimH's pilin domain. Under static conditions, the interaction of the two domains of full-length FimH stabilizes the lectin domain at a low affinity for monomemannose (e.g. K) d300 μ M) state characterized by shallow binding pockets. Binding to the mannoside ligand induces a conformational change resulting in a moderate affinity state in which the lectin is held in close contact with the pilin domain. However, under shear stress, lectins dissociate from the pilin domain, thereby inducing a high affinity state (e.g., K)d<1.2μM)。
The isolated lectin domain of FimH is locked in a high affinity state due to the lack of negative allosteric modulation imposed by the pilin domain. Isolated recombinant lectin domains locked in a high affinity state exhibit high stability. However, locking the adhesin in a low binding conformation induces the production of adhesion-inhibiting antibodies. Therefore, there is interest in stabilizing lectin domains in low affinity states.
There is additional interest in methods to express FimH in high yields sufficient for product development. A barrier to the development of compositions comprising FimH is the low yield achieved when FimH is expressed in its native state in the periplasm of e. Typical yields reported at laboratory scale for purified FimCH complexes are 3-5mg/L, and for fimh (ld) are 4-10mg/L, below the scalable level of clinical trial material production. The in vivo conformation of FimH is different from that obtained by the purified recombinant form of the protein. Generally, FimH has a native conformation that is determined, at least in part, by the in vivo interaction of FimH with its periplasmic chaperone protein (known as FimC).
Recombinant production of FimH remains challenging. Expression and purification of proteins is not a routine procedure.
Summary of The Invention
To meet these and other needs, the present invention is directed to compositions and methods of use thereof for producing recombinant adhesin proteins and for eliciting an immune response against e.
In one aspect, the invention relates to a recombinant mammalian cell comprising a polynucleotide encoding a polypeptide derived from E.coli, or a fragment thereof. In some embodiments, the polynucleotide encodes a polypeptide derived from an e.coli pilus h (fimh) polypeptide or fragment thereof. In some embodiments, the polypeptide derived from e.coli FimH, or fragment thereof, comprises a phenylalanine residue at the N-terminus of the polypeptide.
In one aspect, the invention relates to a method of producing a polypeptide derived from E.coli, or a fragment thereof, in a recombinant mammalian cell. The method comprises culturing a recombinant mammalian cell under suitable conditions such that the polypeptide or fragment thereof is expressed; and harvesting the polypeptide or fragment thereof. In some embodiments, the method further comprises purifying the polypeptide or fragment thereof. In some embodiments, the yield of the polypeptide is at least 0.05 g/L. In some embodiments, the yield of the polypeptide is at least 0.10 g/L.
In one aspect, the invention relates to a composition comprising a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to seq id no:1, 2, 3, 4, 20, 23, 24, 26, 28, 29 or any combination thereof.
In another aspect, the invention relates to a composition comprising at least n consecutive amino acids having any one from the following sequences: 1, 2, 3, 4, 20, 23, 24, 26, 28 and 29, wherein n is 7 or greater (e.g., 8,10,12,14,16,18,20 or greater). In some embodiments, the composition further comprises a saccharide selected from any one of the formulae in table 1 (preferably formula O1A, formula O1B, formula O2, formula O6, and formula O25B), wherein n is an integer from 1 to 100, preferably from 31 to 100.
Drawings
FIG. 1A-FIG. 1H-depict amino acid sequences, including the amino acid sequence of an exemplary polypeptide derived from E.coli, or a fragment thereof; and the amino acid sequence of an exemplary wzzB sequence.
FIGS. 2A-2T-depict maps of exemplary expression vectors.
FIG. 3-depicts the results of expression and purification.
FIG. 4-depicts the results of expression and purification.
FIG. 5-depicts the results of expression.
FIG. 6A-FIG. 6C-depicts the pools and affinities of pSB02083 and pSB02158 SEC; including yield.
Figure 7-depicts the results of pSB2198 FimH dscG lock variant construct expression.
FIG. 8-depicts the results of wild type expression of pSB2307 FimH dscG.
FIGS. 9A-9C-depict the structure of O-antigens synthesized by the polymerase-dependent pathway, which have four or fewer residues in the backbone.
FIG. 10A-FIG. 10B-FIG. 10A depict the structure of an O-antigen synthesized by a polymerase-dependent pathway, which has five or six residues in the backbone; FIG. 10B depicts O-antigens thought to be synthesized by the ABC transporter-dependent pathway.
FIG. 11-depicts a computational mutagenesis scan of Phe1 with other amino acids having aliphatic hydrophobic side chains (e.g., Ile, Leu, and Val) that can stabilize the FimH protein and accommodate mannose binding.
FIG. 12A-FIG. 12B-depict plasmids: pUC replicon plasmid, 500-700 Xcopies per cell, chain regulator (FIG. 12A); and P15a replicon plasmid, 10-12x copies per cell, O-antigen operon (fig. 12B).
FIG. 13A-FIG. 13B-depict the regulation of O-antigen chain length in serotype O25a and O25B strains by plasmid-based expression of heterologous wzzB and fepE chain length regulators. The genetic complementation of LPS expression in plasmid transformants of wzzB knockout strain O25K5H1(O25a) and GAR2401(O25b) is shown. On the left of fig. 13A, the LPS map of plasmid transformants of O25a O25K5H Δ wzzB is shown; the right is a similar pattern for the O25b GAR 2401. delta. wzzB transformant. Immunoblots of replicate gels probed with O25 specific Serum (Statens Serum institute) are shown in FIG. 13B. O25a Δ wxxB (knock-out) background associated with lanes 1-7; o25b 2401. delta. wzzB (knock-out) background associated with lanes 8-15.
FIG. 14-depicts the expression of the long-chain O-antigen conferred by E.coli and Salmonella (Salmonella) fepE plasmids in the host O25K5H1 Δ wzzB.
FIG. 15-depicts Salmonella fepE expression producing the growth O-antigen LPS in various clinical isolates.
FIG. 16A-FIG. 16B-depict plasmid-mediated arabinose-inducible expression of O25B long O-antigen LPS in O25B O-antigen knock-out host strains. The results of the SPS PAGE are shown in fig. 16A and the results of the O25 immunoblot are shown in fig. 16B, where in fig. 16A and 16B, lane 1 is from clone 1, without arabinose; lane 2 from clone 1, 0.2% arabinose; lane 3 from clone 9, no arabinose; lane 4 from clone 9, 0.2% arabinose; lane 5 is from O55 e.coli LPS standard; lane 6 is from O111 E.coli LPS standard.
Figure 17-depicts plasmid-mediated arabinose-inducible expression of the long O antigen LPS in common host strains.
FIG. 18-depicts the expression of O25O-antigen LPS in exploratory bioprocess strains.
FIG. 19A-FIG. 19B-depicts SEC characteristics of short O25B O-antigen (FIG. 19A, strain 1O25B wt 2831) and long O25B O-antigen (FIG. 19B, strain 2O25B 2401 Δ wzB/LT 2 FepE) purified from strain GAR2831 and' 2401 Δ wzB/fepE.
Fig. 20A-fig. 20B-depict rabbit vaccination schedules: (fig. 20A) information on the vaccination schedule of rabbit study 1 VAC-2017-PRL-EC-0723; (FIG. 20B) Rabbit study vaccination schedule of 2 VAC-2018-PRL-EC-077.
FIG. 21A-FIG. 21C-depicts the O25b glycoconjugate IgG response wherein- ● -represents the results prior to exsanguination; - ■ -bleeding 1 st (6 weeks); a-tangle-solidup-2 bleeding (8 weeks); -. 3 rd bleeding (12 weeks). FIG. 21A depicts results from rabbits 1-3 (media activation); FIG. 21B depicts results from rabbits 2-3 (low activation); FIG. 21C depicts results from rabbit 3-1 (highly activated).
FIG. 22A-FIG. 22F-depicts long O-antigen glycoconjugates to O25b, i.e., low activation O25b-CRM197The IgG response of the conjugate (FIG. 22D-FIG. 22F, where- ● -represents pre-exsanguination results from rabbit 2-1, - ■ -represents week 12 antisera results from rabbit 2-1) versus the unconjugated polysaccharide, free O25b polysaccharide (FIG. 22A-FIG. 22C, where- ● -represents pre-exsanguination results from rabbit A-1, - ■ -represents week 6 antisera results from rabbit A-1, -tangle-solidup-represents week 8 antisera results from rabbit A-1). Note that MFI is plotted on a logarithmic scale to emphasize the difference between pre-immune and immune antibodies <Difference in the range of 1000 MFI. FIG. 22A depicts results from rabbit A-1 (unconjugated polysaccharide); FIG. 22B depicts results from rabbit A-3 (unconjugated polysaccharide); FIG. 22C depicts results from rabbit A-4 (unconjugated polysaccharide); FIG. 22D depicts results from rabbit 2-1 (low activation); FIG. 22E depicts the results of rabbit 2-2 (low degree of activation); and FIG. 22F depicts nodules from rabbit 2-3 (low activation)And (5) fruit.
FIG. 23A-FIG. 23C-depict surface expression of native versus long O25b O-antigen as detected with O25b antiserum. FIG. 23A depicts the results, where- ● -represents the results from O25b2831 control PD3 antisera; - ■ -represents the result of O25b2831 wt versus pre-exsanguination; a-represents the result of O25b2831/fepE vs PD3 antisera;
Figure BDA0003622971360000061
represents the result of O25b2831/fepE compared with the exsanguination. FIG. 23B depicts results, where- ● -represents results from O25B 2401 vs PD3 antiserum; - ■ -represents the result of O25b 2401 versus before exsanguination; a-represents the result of O25b 2401/fepE vs PD3 antiserum;
Figure BDA0003622971360000062
represents the results of O25b 2401/fepE comparison before exsanguination. FIG. 23C depicts the results, wherein- ● -represents results from E.coli K12 versus PD3 antisera; and- ■ -represents the results of E.coli K12 versus before exsanguination.
Figure 24-depicts the general structure of the carbohydrate backbone of five exo-oligosaccharides of known chemical type. All sugars are in the alpha anomeric configuration unless otherwise indicated. The genes whose products catalyze the formation of each linkage are indicated by dashed arrows. Asterisks indicate the residues of the core oligosaccharide where O-antigen attachment occurs.
Figure 25-depicts the lack of immunogenicity (dLIA) of unconjugated free O25b polysaccharide, wherein- ● -represents the results from week 18 (1wk ═ PD4) antisera from 4-1; representative- ■ -representative of results from week 18 (1wk — PD4) antiserum from 4-2; a-represents the results from week 18 (1wk ═ PD4) antisera from 5-1;
Figure BDA0003622971360000063
represents the results from week 18 (1 wk-PD 4) antisera from 5-2;
Figure BDA0003622971360000064
represents the results from week 18 (1 wk-PD 4) antisera from 6-1;
Figure BDA0003622971360000065
represents the results from week 18 (1 wk-PD 4) antisera from 6-2.
FIGS. 26A-26C-depict graphs demonstrating the specificity of immune serum OPA titers of BRC rabbit O25b RAC conjugate. FIG. 26A shows the OPA titers of pre-immune serum (- ● -) and post-immune serum (- ■ -) for rabbit 2-3 at week 13. FIG. 26B shows the OPA titers of pre-immunization serum (- ● -) and post-19-week-immunization serum (- ■ -) in rabbits 1-2. FIG. 26C shows the specificity of the OPA titer of rabbit 1-2 at week 19, where the OPA activity of rabbit 1-2 immune serum was blocked by preincubation with 100. mu.g/mL of purified unconjugated O25b long O-antigen polysaccharide, where- ■ -represents the results at week 19 of rabbit 1-2 immune serum;
Figure BDA0003622971360000071
Represents the results of rabbit 1-2 weeks 19/R1 long-OAg.
Figures 27A-27C-27A depict diagrams of exemplary administration schedules. FIGS. 27B and 27C show a schematic depicting the conjugation of unconjugated O25B long O-antigen polysaccharide (FIG. 27B, O25B free polysaccharide (2 μ g)) and derivatized O25B RAC/DMSO long O-antigen glycoconjugates (FIG. 27C, O25B-CRM)197RAC long (2 μ g)) elicited O-antigen O25b IgG levels, where- (dashed line) represents the original CD 1O 25b IgG levels.
Figure 28A-figure 28B-depicts graphs showing OPA immunogenicity of RAC, eTEC O25B long glycoconjugates and single-ended glycoconjugates after 2 nd (figure 28A) and after 3 rd (figure 28B) dosing, wherein-O-represents results from a single-ended short circuit of 2 μ g; - ● -represents a result with a single end length of 2 μ g; a-represents results with RAC/DMSO length 2 μ g;
Figure BDA0003622971360000072
represents the results for eTEC at 2. mu.g length; background control (n-20).
Figure BDA0003622971360000074
The responder ratio is as follows>2-fold% of mice not vaccinated at baseline titers.
FIG. 29-depictsA graph showing altered levels of OPA immunogenicity and polysaccharide activation for eTEC chemistry.
Figure BDA0003622971360000075
The responder ratio is as follows>2-fold% of mice with non-vaccinated baseline titers.
Figure 30A-figure 30B-diagrams depicting exemplary administration schedules (figure 30A); and describes protection of mice immunized with multiple doses of e.coli eTEC conjugate from lethal challenge with O25B isolate (fig. 30B), where-represents 17% activation of the eTEC long chain; -. DELTA-eTEC for 10% activation of the long chain;
Figure BDA0003622971360000073
Representing 4% activation of eTEC long chains; - □ -represents a polysaccharide O25 b; -. O-represents an uninoculated control.
Figure 31-depicts a schematic illustrating an exemplary preparation of a single-ended conjugate, wherein the conjugation process involves selective activation of 2-keto-3-deoxyoctanoic acid (KDO) with an amine disulfide linker upon exposure of the thiol functional group. KDO is then reacted with bromine-activated CRM197Protein conjugation, as shown in figure 31 (preparation of single-ended conjugates).
FIGS. 32A-32B-depict methods for preparation and CRM197Exemplary process flow diagrams for activation (fig. 32A) and conjugation (fig. 32B) of e.
Sequence identifier
SEQ ID NO. 1 shows the amino acid sequence of the wild type 1 pilus D-mannose specific adhesin [ E.coli FimH J96 ].
The amino acid sequence of the FimH fragment is given in SEQ ID NO:2, corresponding to aa residues 22-300 of SEQ ID NO:1 (mature FimH protein).
The amino acid sequence of the FimH lectin domain is given in SEQ ID NO 3.
The amino acid sequence of the FimH pilin domain is given in SEQ ID No. 4.
SEQ ID NO:5 gives the amino acid sequence of the polypeptide derived from E.coli fimH (fimH mIgK signal peptide in pSB02198-pcDNA3.1 (+)/F22.. Q300J 96fimH N28S V48C L55C N91S N249Q/7AA linker/fimG A1.. K14/GGHis8)
SEQ ID NO 6 shows the amino acid sequence of the polypeptide derived from Escherichia coli fimH (fimH mIgK signal peptide in pSB02307-pcDNA3.1 (+)/F22.. Q300J 96fimH N28S N91S N249Q/His8)
SEQ ID NO 7 shows the amino acid sequence of a polypeptide fragment derived from E.coli FimH (pSB02083 FimH lectin domain wild-type construct)
SEQ ID NO 8 shows the amino acid sequence of a polypeptide fragment derived from Escherichia coli FimH (pSB02158 FimH lectin domain locked mutant)
SEQ ID NO 9 shows the amino acid sequence of a polypeptide fragment derived from Escherichia coli FimG (FimG A1.. K14)
The amino acid sequence of the polypeptide fragment derived from E.coli FimC is given in SEQ ID NO 10.
SEQ ID NO 11 gives the amino acid sequence of the 4aa linker.
SEQ ID NO 12 gives the amino acid sequence of the 5aa linker.
SEQ ID NO 13 gives the amino acid sequence of the 6aa linker.
SEQ ID NO 14 gives the amino acid sequence of the 7aa linker.
SEQ ID NO 15 gives the amino acid sequence of the 8aa linker.
SEQ ID NO 16 gives the amino acid sequence of the 9aa linker.
SEQ ID NO 17 gives the amino acid sequence of the 10aa linker.
18 amino acid sequence of the FimH J96 signal sequence
SEQ ID NO 19 gives the amino acid sequence of the signal peptide of SEQ ID NO 5 (fimH mIgK signal peptide/F22.. Q300J 96fimH N28S V48C L55C N91S N249Q/7AA linker/fimG A1.. K14/GGHis8 in pSB02198-pcDNA3.1 (+).
SEQ ID NO 20 shows the fimH mIgK signal peptide/F22.. Q300J96 fimH N28S V48C L55C N91S N249Q/7AA linker/fimG A1.. K14/GGHis8 in accordance with SEQ ID NO 5 (mature protein of pSB 02198-pcDNA3.1 (+).
SEQ ID NO 21 shows the amino acid sequence of a polypeptide derived from E.coli FimG.
The amino acid sequence of the signal peptide of SEQ ID NO. 6 is given in SEQ ID NO. 22 (FimH mIgK signal peptide/F22.. Q300J96 FimH N28S N91S N249Q/His8 in pSB02307-pcDNA3.1 (+).
The amino acid sequence of the polypeptide derived from E.coli fimH according to SEQ ID NO 6 is given in SEQ ID NO 23 (fimH mIgK signal peptide of pcDNA3.1 (+)/F22.. Q300J96 fimH N28S N91S N249Q/His8 mature protein).
The amino acid sequence of the polypeptide of FimH derived from escherichia coli (mature protein of the pSB02083 FimH lectin domain wild-type construct) according to SEQ ID No. 7 is given in SEQ ID No. 24.
The amino acid sequence of the His tag is given in SEQ ID NO 25.
SEQ ID NO 26 shows the amino acid sequence of a polypeptide derived from Escherichia coli FimH according to SEQ ID NO 8 (mature protein of pSB02158 FimH lectin domain knob)
The amino acid sequence of a polypeptide derived from E.coli FimH (pSB01878) is given in SEQ ID NO 27.
The amino acid sequence of the polypeptide from E.coli FimH (K12) is given in SEQ ID NO 28.
SEQ ID NO 29 gives the amino acid sequence of the polypeptide derived from E.coli FimH (UTI 89).
The O25b 2401WzzB amino acid sequence is given in SEQ ID NO 30.
The amino acid sequence O25a: K5: H1 WzzB is given in SEQ ID NO 31.
The O25a ETEC ATCC WzzB amino acid sequence is given in SEQ ID NO. 32.
The amino acid sequence K12W 3110 WzzB is given in SEQ ID NO 33.
The Salmonella LT2WzzB amino acid sequence is given in SEQ ID NO 34.
The amino acid sequence of O25b 2401FepE is given in SEQ ID NO 35.
The amino acid sequence of O25a: K5: H1 FepE is given in SEQ ID NO 36.
The O25a ETEC ATCC FepE amino acid sequence is given in SEQ ID NO 37.
The amino acid sequence of O157 FepE is given in SEQ ID NO 38.
The amino acid sequence of Salmonella LT2 FepE is given in SEQ ID NO 39.
The primer sequence for LT2wzZB _ S is given in SEQ ID NO 40.
The primer sequence for LT2 wzB _ AS is given in SEQ ID NO 41.
The primer sequence for O25bFepE _ S is given in SEQ ID NO 42.
The primer sequence for O25bFepE _ A is given in SEQ ID NO 43.
The primer sequence of wzB P1_ S is given in SEQ ID NO. 44.
The primer sequence of wzB P2_ AS is given in SEQ ID NO. 45.
The primer sequence of wzB P3_ S is given in SEQ ID NO 46.
The primer sequence of wzZB P4_ AS is given in SEQ ID NO. 47.
The primer sequence for O157 FepE _ S is given in SEQ ID NO 48.
The primer sequence for O157 FepE _ AS is given in SEQ ID NO 49.
The primer sequence of pBAD33_ adapter _ S is given in SEQ ID NO. 50.
The primer sequence of pBAD33_ adapter _ AS is given in SEQ ID NO. 51.
SEQ ID NO 52 shows the primer sequence of JUMPSTART _ r.
The primer sequence of gnd _ f is given in SEQ ID NO 53.
The amino acid sequence of the mouse IgK signal sequence is given in SEQ ID NO 54.
The amino acid sequence of the signal peptide p51 of the large subunit of FcRn of the human IgG receptor is shown in SEQ ID NO. 55.
The amino acid sequence of the signal peptide of human IL10 protein is given in SEQ ID NO. 56.
The amino acid sequence of the fusion glycoprotein F0 signal peptide of human respiratory syncytial virus type A (strain A2) is given in SEQ ID NO. 57.
The amino acid sequence of the influenza A hemagglutinin signal peptide is given in SEQ ID NO 58.
SEQ ID NOS 59-101 show the amino acid and nucleic acid sequences of nanostructure-related polypeptides or fragments thereof.
102-109 gave the SignalP 4.1(DTU Bioinformatics) sequence of the various species used for signal peptide prediction.
Disclosure of Invention
The present inventors overcome the challenge of producing polypeptides derived from E.coli adhesin proteins by using mammalian cells for expression. As illustrated throughout the present disclosure and in the examples section, it was found that mammalian cell expression of recombinant polypeptides consistently results in high yields as compared to expression of the polypeptide in E.coli. In addition, the present inventors have surprisingly identified mutation and expression constructs to stabilize recombinant polypeptides and fragments thereof in a desired conformation.
Blocking the primary stage of infection (i.e., bacterial attachment to host cell receptors and colonization of mucosal surfaces) is important for preventing, treating and/or reducing the likelihood of bacterial infection. Bacterial attachment may involve the interaction of bacterial surface proteins, known as adhesins, with host cell receptors. Previous preclinical studies on FimH adhesin (from uropathogenic e.coli) have demonstrated that antibodies against adhesin can be elicited. To prevent infections from otitis media and caries to pneumonia and sepsis, advances in the identification, characterization and isolation of adhesins are needed.
In order to produce adhesin proteins such as FimH and fragments thereof on a commercial scale, it is necessary to identify suitable constructs and suitable hosts so that the polypeptides and fragments thereof can be expressed in sufficient amounts and in a preferred conformation over a sustained period of time. For example, in some embodiments, the preferred conformation of the recombinant polypeptide exhibits low affinity for monomemannose (e.g., K)d300. mu.M). In some embodiments, the preferred conformation exhibits high affinity for monomemannose (e.g., K)d<1.2μM)。
Adhesin proteins from E.coli have been expressed recombinantly in E.coli cells. However, the yield has been below 10 mg/L. Purification of large quantities of pilus-associated adhesins can be challenging when produced in e. Without being bound by theory or mechanism, it is believed that the product expressed in E.coli may exhibit a conformation that is not optimal for eliciting an effective immune response in a mammal.
In one aspect, the invention includes a recombinant mammalian cell comprising a polynucleotide sequence encoding a polypeptide derived from a bacterial adhesin protein, or a fragment thereof.
In another aspect, the invention includes a method for producing a polypeptide or fragment thereof in a mammalian cell, comprising: (i) culturing a mammalian cell under suitable conditions to express the polypeptide or fragment thereof; and (ii) harvesting the polypeptide or fragment thereof from the culture. The method may further comprise purifying the polypeptide or fragment thereof. Also disclosed herein are polypeptides or fragments thereof produced by the methods.
In another aspect, the invention includes a composition comprising a polypeptide described herein or a fragment thereof. The composition may comprise a polypeptide or fragment thereof suitable for in vivo administration. For example, the polypeptide or fragment thereof in such compositions can have a purity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by mass. The composition may further comprise an adjuvant.
In another aspect, the invention includes a composition for inducing an immune response against e. Also disclosed is the use of a composition described herein for inducing an immune response against E.coli, and the use of a composition described herein for the manufacture of a medicament for inducing an immune response against E.coli.
I. Polypeptides derived from Escherichia coli and fragments thereof
In one aspect, disclosed herein is a mammalian cell comprising a polynucleotide encoding a polypeptide derived from escherichia coli, or a fragment thereof. As used herein, the term "derived from" refers to a polypeptide comprising the amino acid sequence of a FimH polypeptide or FimCH polypeptide complex or fragment thereof described herein, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. Preferably, the polypeptide derived from e.coli or fragment thereof comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the corresponding wild-type e.coli FimH polypeptide or fragment thereof. In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, has the same total amino acid length as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex, or fragment thereof.
The fragment should include at least n contiguous amino acids from the sequence, and depending on the particular sequence, n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the fragment comprises an amino acid sequence of at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of the polypeptide derived from e.
In some embodiments, a polypeptide derived from e.coli, or fragment thereof, comprises one or more non-canonical amino acids as compared to a corresponding wild-type e.coli FimH polypeptide or fragment.
In some embodiments, the polypeptide derived from e.coli, or fragment thereof, has a similar or identical function to the corresponding wild-type FimH polypeptide, or fragment thereof.
In a preferred embodiment, the polypeptide or polypeptide complex of the invention or a fragment thereof is isolated or purified.
In some embodiments, the polynucleotide encoding the e.coli-derived polypeptide or fragment thereof is integrated into the genomic DNA of the mammalian cell and the e.coli-derived polypeptide or fragment thereof is expressed by the mammalian cell when cultured under suitable conditions.
In a preferred embodiment, the polypeptide derived from E.coli or a fragment thereof is soluble.
In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, is secreted from a mammalian host cell.
In some embodiments, the polypeptide derived from e.coli, or fragment thereof, may comprise additional amino acid residues, such as an N-terminal or C-terminal extension. Such extensions may comprise one or more tags that may facilitate detection (e.g., epitope tags for detection by monoclonal antibodies) and/or purification (e.g., polyhistidine tags that allow purification on nickel chelating resins) of the polypeptide or fragment thereof. In some embodiments, the tag comprises an amino acid sequence selected from any one of SEQ ID NO:21 and SEQ ID NO: 25. Such affinity purification tags are known in the art. Examples of affinity purification tags include e.g. a His-tag (hexa-histidine, which may e.g. be bound to a metal ion), Maltose Binding Protein (MBP), which may e.g. be bound to amylose, glutathione-S-transferase (GST), which may e.g. be bound to glutathione, a FLAG-tag, which may e.g. be bound to an anti-FLAG antibody, a Strep-tag, which may e.g. be bound to streptavidin or a derivative thereof. In a preferred embodiment, the polypeptide derived from E.coli or a fragment thereof does not comprise additional amino acid residues, such as an N-terminal or C-terminal extension. In some embodiments, the polypeptides derived from e.coli described herein, or fragments thereof, do not comprise an exogenous tag sequence.
Although specific strains of E.coli may be referred to herein, it should be understood that polypeptides derived from E.coli, or fragments thereof, are not limited to a particular strain unless otherwise specified.
In some embodiments, the polypeptide derived from e.coli FimH, or fragment thereof, comprises a phenylalanine residue at the N-terminus of the polypeptide. In some embodiments, the FimH-derived polypeptide or fragment thereof comprises a phenylalanine residue within the first 20 residue positions of the N-terminus. Preferably, the phenylalanine residue is located at position 1 of the polypeptide. For example, in some embodiments, the polypeptide derived from e.coli FimH or fragment thereof does not comprise an additional glycine residue at the N-terminus of the polypeptide derived from e.coli FimH or fragment thereof.
In some embodiments, the phenylalanine residue at position 1 of the wild-type mature e.coli FimH is replaced with an aliphatic hydrophobic amino acid (e.g., any of the Ile, Leu, and Val residues).
In some embodiments, the signal peptide can be used to express a polypeptide derived from E.coli, or a fragment thereof. Signal sequences and expression cassettes for the production of proteins are known in the art. Typically, leader peptides are 5-30 amino acids in length and are usually present at the N-terminus of the newly synthesized polypeptide. Signal peptides typically comprise a long stretch of hydrophobic amino acids that tend to form a single alpha-helix. In addition, many signal peptides start with a small stretch of positively charged amino acids that can help to enhance the correct topology of the polypeptide during translocation. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during or after translocation is complete, generating a free signal peptide and a mature protein. In some embodiments, the signal peptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of the following sequences: 9, 18, 19 and 22.
In some embodiments, the polypeptides derived from e.coli described herein or fragments thereof may comprise a cleavable linker. Such linkers allow the tag to be separated from the purified complex, for example by the addition of an agent capable of cleaving the linker. Cleavable linkers are known in the art. Such linkers may be cleaved, for example, by irradiation of a photolabile bond or acid catalyzed hydrolysis. Another example of a cleavable linker includes a polypeptide linker that incorporates a protease recognition site and can be cleaved by the addition of a suitable protease.
In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises a modification as compared to a corresponding wild-type escherichia coli FimH polypeptide or fragment. The modification may comprise covalent attachment of the molecule to the polypeptide. For example, such modifications may include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. In some embodiments, the e.coli-derived polypeptide or fragment thereof may comprise modifications, such as chemical modifications by using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like, as compared to the corresponding wild-type e.coli FimH polypeptide or fragment. In another embodiment, the modification may comprise covalent attachment of a lipid molecule to the polypeptide. In some embodiments, the polypeptide does not comprise a molecular covalent linkage to the polypeptide, as compared to a corresponding wild-type e.
For example, proteins and polypeptides produced in cell culture can be glycoproteins that contain covalently linked carbohydrate structures including oligosaccharide chains. These oligosaccharide chains are linked to the protein by an N-linkage or an O-linkage. The oligosaccharide chains may comprise a substantial proportion of the mass of the glycoprotein. Typically, the N-linked oligosaccharide is added to the amino group of the side chain of an asparagine residue within the target consensus sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. In some embodiments, the glycosylation site comprises an amino acid sequence selected from any one of the following sequences: asparagine-glycine-threonine (NGT), asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS). The polypeptide derived from E.coli or a fragment thereof produced in mammalian cells may be glycosylated. Glycosylation can occur at the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the sequence of a polypeptide derived from E.coli or a fragment thereof. By "N-linked glycosylation" is meant that the carbohydrate moiety is attached to an asparagine residue in a polypeptide chain through GIcNAc. The N-linked carbohydrate contains the common Man 1-6(Man1-3) Man β 1-4GlcNAc β 1-4GlcNAc β -R core structure, where R represents the asparagine residue of the resulting polypeptide derived from E.coli or a fragment thereof.
In some embodiments, the glycosylation site in the e.coli-derived polypeptide or fragment thereof is removed by mutation within the sequence of the e.coli-derived polypeptide or fragment thereof. For example, in some embodiments, it may be preferable to mutate the Asn residue of the glycosylation motif (Asn-Xaa-Ser/Thr) by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and gin.
In some embodiments, the Ser residue of the glycosylation motif can be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and gin.
In some embodiments, it may be preferred to mutate the Thr residue of the glycosylation motif by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and gin.
In some embodiments, the glycosylation site (such as Asn-Xaa-Ser/Thr) in the polypeptide derived from E.coli or fragment thereof is not removed or modified. In some embodiments, compounds that reduce or inhibit glycosylation can be added to the cell culture medium. In such embodiments, the polypeptide or protein comprises at least one more non-glycosylated (i.e., non-glycosylated) site (i.e., a completely unoccupied glycan site to which no carbohydrate moiety is attached) or at least one less carbohydrate moiety at the same potential glycosylation site as compared to an otherwise identical polypeptide or protein produced by the cell under otherwise identical conditions but in the absence of the glycosylation inhibiting compound. These compounds are known in the art and may include, but are not limited to, tunicamycin homologues, streptoviridins, brazidomycin, amfomycin, ziromycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynebacteriaxin, pyrophyllomycin, duocarmycin (duimycin), 1-deoxymannonojirimycin (1-deoxymannonojirimycin), deoxynojirimycin, N-methyl-1-deoxymannojirimycin, brefeldin A, glucose and mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D- (+) -mannose, D- (+) galactose, 2-deoxy-2-fluoro-D-glucose, 1, 4-dideoxy-1, 4-imino-D-mannitol (DIM), Fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl-CoA reductase inhibitor, 25-hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, methylcarbonyl cyanophenylhydrazone (CCCP), compactin, polyterpenyl-phosphoryl-2-deoxyglucose (dolichyl-deoxyglucide), N-acetyl-D-glucosamine, isoxanthine (hygroxanthine), thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromconditol, condulcitol epoxide and condulcitol derivatives, glycosylmethyl-p-yltrivazalene, beta-hydroxy-noralanine, threo-beta-fluoroaspartamide, beta-fluoroglutaramide, beta-hydroxyvaleronitrile, beta-deoxyglucose, beta-glucosidase, beta-deoxyglucose, and pharmaceutically acceptable salts thereof, D- (+) -glucono delta-lactone, di (2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (diphenylmethyl) -phosphate, [2- (diphenylphosphonoyloxy) ethyl ] trimethylammonium iodide, iodoacetate and/or fluoroacetate. One of ordinary skill in the art will readily recognize or be able to determine glycosylation inhibiting materials that can be used in accordance with the methods and compositions of the present invention without undue experimentation. In such embodiments, glycosylation of the polypeptide or fragment thereof can be controlled without introducing amino acid mutations in the polypeptide or fragment thereof.
In some embodiments, the level of glycosylation (e.g., the number of glycan sites occupied on the polypeptide or fragment thereof, the size and/or complexity of the glycoform at such sites, etc.) of the polypeptide or fragment thereof produced by the mammalian cell is lower than the level of glycosylation of the polypeptide or fragment thereof produced under otherwise identical conditions in otherwise identical media lacking such glycolysis inhibition compounds and/or mutations.
In some embodiments, the sequence of the E.coli-derived polypeptide or fragment thereof does not include N-linked protein glycosylation sites. In some embodiments, the sequence of the E.coli-derived polypeptide or fragment thereof does not include at least one N-linked protein glycosylation site. In some embodiments, the sequence of the E.coli-derived polypeptide or fragment thereof does not include any N-linked protein glycosylation sites. In some embodiments, the sequence of the polypeptide derived from E.coli or fragment thereof includes an N-linked protein glycosylation site. In some embodiments, the sequence of the E.coli-derived polypeptide or fragment thereof comprises up to 1N-linked glycosylation site of the protein. In some embodiments, the sequence of the E.coli-derived polypeptide or fragment thereof comprises up to 2N-linked protein glycosylation sites.
The e.coli-derived polypeptides or fragments thereof expressed in different cell lines or transgenic animals may have different glycan site occupancy, glycoform and/or glycosylation patterns compared to each other. In some embodiments, the invention encompasses polypeptides derived from e.coli or fragments thereof, regardless of the glycosylation, glycan occupancy, or glycoform pattern of the e.coli-derived polypeptides or fragments thereof produced in the mammalian cell.
In some embodiments, the E.coli-derived polypeptide or fragment thereof can be derived from an E.coli FimH polypeptide in which the amino acid residue at position 1 of the polypeptide is phenylalanine, rather than methionine, e.g., a polypeptide having the amino acid sequence of SEQ ID NO. 2. Preferably, the polypeptide derived from escherichia coli FimH comprises phenylalanine at position 1 of the amino acid sequence of the polypeptide derived from escherichia coli. In another preferred embodiment, the polypeptide derived from E.coli FimH comprises the amino acid sequence SEQ ID NO 3, preferably wherein the residue at position 1 of the amino acid sequence of the polypeptide derived from E.coli is phenylalanine. In some embodiments, the E.coli-derived polypeptide or fragment thereof can include the amino acid sequence SEQ ID NO 4, which can be derived from an E.coli FimH polypeptide.
In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of the following sequences: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, 24, 26, 27, 28 and 29. In some embodiments, the polypeptide derived from E.coli, or fragment thereof, can be derived from an E.coli FimG polypeptide, e.g., having the amino acid sequence of SEQ ID NO 9. In some embodiments, the polypeptide derived from e.coli, or fragment thereof, may be derived from e.coli FimC polypeptide, e.g., having the amino acid sequence SEQ ID NO: 10.
A. Polypeptides derived from escherichia coli FimH and fragments thereof
In a preferred embodiment, the polypeptide or fragment thereof is derived from E.coli FimH. In some embodiments, the polypeptide or fragment thereof comprises full-length e. Full-length FimH includes two domains: an N-terminal lectin domain and a C-terminal pilin domain, connected by a short linker. In some embodiments, the full length of e.coli FimH comprises 279 amino acids, which includes the full length of the mature protein of e.coli FimH. In some embodiments, the full length of e.coli FimH comprises 300 amino acids, which includes the full length of the mature protein of e.coli FimH and a signal peptide sequence that is 21 amino acids in length. The primary structure of the 300 amino acid long wild-type FimH is highly conserved among strains of e.
An exemplary sequence of full-length E.coli FimH is SEQ ID NO 1. The full-length FimH sequence includes the sequence of the lectin domain and the sequence of the pilin domain. The lectin domain of FimH comprises a carbohydrate recognition domain that is responsible for binding to mannosylated urolytic protein 1a on the surface of urothelial cells. The pilin domain is anchored to the core of the pilus by the donor chain of the subsequent FimG subunit, a process called donor chain complementation.
Starting from the N-terminus, the names of each domain of full-length FimH and exemplary amino acid sequences in parentheses are as follows: FimH lectin (SEQ ID NO:2) and FimH pilin (SEQ ID NO: 3).
Other suitable polypeptides and fragments thereof derived from e.coli FimH include variants with varying degrees of identity to any one of the following sequences: 1, 2, 3, 4, 20, 23, 24, 26, 28, 29, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to any of the following sequences: 1, 2, 3, 4, 20, 23, 24, 26, 28 and 29. In certain embodiments, the FimH variant protein: (i) forming a part of FimH-FimC; (ii) comprises at least one epitope from SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28 and SEQ ID NO 29; and/or (iii) an antibody that elicits an immunological cross-reaction with E.coli FimH in vivo.
In some embodiments, the composition comprises a polypeptide having at least n consecutive amino acids from any one of the following sequences: 1, 2, 3, 4, 20, 23, 24, 26, 28 and 29, wherein n is 7 or greater (e.g., 8,10,12,14,16,18,20 or greater). Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of any one of: 1, 2, 3, 4, 20, 23, 24, 26, 28 and 29.
In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 1. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 2. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 3. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 4. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 20. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 23. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 24. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 26. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 28. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 30.
Another example of a suitable polypeptide derived from E.coli FimH and fragments thereof as described herein is shown in SEQ ID NO. 2, which lacks the wild-type N-terminal signal sequence and corresponds to amino acid residues 22-300 of SEQ ID NO. 1. Another example of a FimH fragment comprises the entire N-terminal signal sequence and the mature protein, such as shown in SEQ ID NO: 1.
In some embodiments, the glycosylation site in the polypeptide derived from E.coli or fragment thereof is removed by mutation in the sequence of the polypeptide derived from E.coli or fragment thereof. For example, in some embodiments, it may be preferred to mutate the Asn residue at position 7 (e.g., numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, it may be preferred to mutate the Asn residue at position 7 (e.g.numbering according to SEQ ID NO: 3) of the lectin domain of the E.coli FimH polypeptide by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and gin.
In some embodiments, it may be preferred to mutate the Thr residue at position 10 (e.g., numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, it may be preferred to mutate the Thr residue at position 7 (e.g.numbering according to SEQ ID NO: 3) of the lectin domain of the E.coli FimH polypeptide by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and gin.
In some embodiments, it may be preferred to mutate the Asn residue at position N235 (e.g.numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, it may be preferred to mutate the Asn residue at position N228 (e.g.numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and gin.
In some embodiments, it may be preferred to mutate the Asn residue at position 70 (e.g.numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, it may be preferred to mutate the Asn residue at position 70 (e.g., numbering according to SEQ ID NO: 3) of the lectin domain of an E.coli FimH polypeptide by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and gin.
In some embodiments, it may be preferred to mutate the Ser residue at position 72 (e.g.numbering according to SEQ ID NO: 2) of the mature E.coli FimH polypeptide by substitution. In some embodiments, it may be preferred to mutate the Ser residue at position 72 (e.g.numbering according to SEQ ID NO: 3) of the lectin domain of the E.coli FimH polypeptide by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and gin.
The term "fragment" as used herein refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or has the characteristics of that polypeptide. As used herein, the term also refers to any discrete portion of a given polypeptide that retains at least a portion of the activity of the full-length polypeptide. In certain embodiments, the retained active portion is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the active portion retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the full-length polypeptide. In certain embodiments, the active portion retained is at least 95%, 96%, 97%, 98% or 99% of the activity of the full-length polypeptide. In certain embodiments, the remaining active portion is 100% or more of the activity of the full-length polypeptide. In some embodiments, a fragment includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more contiguous amino acids of the full-length polypeptide.
Complexes of fimh, FimC and fragments thereof
In some embodiments, the polypeptide derived from e.coli FimH or fragment thereof and the polypeptide derived from e.coli FimC or fragment thereof are present in a complex. In a preferred embodiment, the polypeptide derived from e.coli FimH or a fragment thereof and the polypeptide derived from e.coli FimC or a fragment thereof are present in the complex, preferably in a ratio of 1: 1. Without being bound by theory or mechanism, full-length FimH can be stabilized in the active conformation by a periplasmic chaperone (FimC), thus making purification of the full-length FimH protein possible. Thus, in some embodiments, the polypeptide or fragment thereof comprises full-length FimH and full-length FimC.
In some embodiments, the polypeptide or fragment thereof comprises a fragment of FimH and a fragment of FimC. In some embodiments, the polypeptide or fragment thereof comprises a fragment of full-length FimH and FimC. An exemplary sequence of E.coli FimC is shown in SEQ ID NO 10. In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises a complex-forming fragment of FimH.
A complex-forming fragment of FimH may be any part or portion of a FimH protein that retains the ability to form a complex with FimC or a fragment thereof. Suitable complex-forming fragments of FimH may also be obtained or determined by standard assays known in the art such as co-immunoprecipitation assays, cross-linking or fluorescent staining co-localization, etc., as well as by SDS-PAGE or western blot (e.g., by showing FimH fragments and FimC or fragments thereof in a complex as evidenced by gel electrophoresis). In certain embodiments, the complex-forming fragment of FimH (i) forms part of a FimH-FimC complex; (ii) comprising at least one epitope from the following sequences: 1, 2, 3, 4, 10, 20, 23, 24, 26, 28 and 29; and/or (iii) antibodies that elicit an immunological cross-reaction with E.coli FimH in vivo.
In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises full-length FimH, wherein FimH is not complexed with FimC. In yet another embodiment, the polypeptide or fragment thereof comprises a fragment of FimH, wherein the fragment is not complexed with FimC. In some embodiments, the polypeptide derived from E.coli FimC or a fragment thereof comprises the sequence set forth in SEQ ID NO 10. In some embodiments, the complexes may be expressed from the same plasmid, preferably under the control of separate promoters for each polypeptide or fragment thereof.
In some embodiments, the polypeptide derived from e.coli FimH or fragment thereof is combined with a polypeptide derived from e.coli FimC or fragment thereof, which can be engineered into the structure of the polypeptide derived from e.coli FimH or fragment thereof. The portion of the FimC molecule that binds to FimH in the complex is called the "donor strand" and the mechanism of forming the native FimH structure using the FimC-binding strand of the FimH complex is called "donor strand complementation".
In some embodiments, a polypeptide derived from e.coli FimH, or a fragment thereof, can be expressed from a suitable donor strand complementary form of FimH, wherein the amino acid sequence of FimC that interacts with FimH in the FimCH complex is itself engineered at the C-terminus of FimH to provide the native conformation, without the presence of the remainder of the FimC molecule. In some embodiments, the polypeptide derived from e.coli FimH, or a fragment thereof, may be expressed in the form of a complex comprising its isolated domains, such as a lectin-binding domain and a pilin domain, and such domains may be linked together covalently or non-covalently. For example, in some embodiments, the linking segment can include an amino acid sequence or other oligomeric structure, including simple multimeric polymeric structures.
The methods and compositions of the invention may include complexes as described herein, wherein the polypeptides derived from E.coli, or fragments thereof, are co-expressed or formed in a combined state.
C. Lectin domains, pilin domains and variants thereof
The conformational and ligand binding properties of FimH's lectin domain may be under allosteric control of FimH's pilin domain. Under static conditions, the interaction of the two domains of full-length FimH stabilizes the lectin domain against monomemannoseLow affinity state (e.g. K)d300 μ M) characterized by shallow binding pockets. Binding to the mannoside ligand can induce a conformational change, resulting in a moderate affinity state in which the lectin and pilin domains are held in intimate contact. However, under shear stress, lectin and pilin domains can separate and induce a high affinity state (e.g., K)d<1.2μM)。
The isolated lectin domain of FimH is locked in a high affinity state (e.g., K) due to the lack of negative allosteric modulation by the pilin domaind<1.2. mu.M). An isolated recombinant lectin domain locked in a high affinity state. However, the adhesins are locked in a low affinity conformation (e.g. K) d300. mu.M) induces the production of adhesion-inhibiting antibodies. Therefore, there is interest in stabilizing lectin domains in low affinity states.
In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises the lectin domain of escherichia coli FimH. Exemplary sequences of lectin domains include any of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 24 and SEQ ID NO 26. In some embodiments, the lectin domain of e.coli FimH comprises a cysteine substitution. In a preferred embodiment, the lectin domain of E.coli FimH comprises a cysteine substitution within the first 50 amino acid residues of the lectin domain. In some embodiments, a lectin domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine substitutions. Preferably, the lectin domain comprises 2 cysteine substitutions. See, for example, pSB02158 and pSB 02198.
Other suitable polypeptides derived from E.coli FimH and fragments thereof include variants having varying degrees of identity to SEQ ID NO. 3, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO. 3. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 3. In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises the pilin domain of escherichia coli FimH. Other suitable polypeptides derived from E.coli FimH and fragments thereof include FimH pilin domain variants having varying degrees of identity to SEQ ID NO. 7, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO. 7. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 4. Other suitable polypeptides derived from E.coli FimH and fragments thereof include FimH lectin domain variants having varying degrees of identity to SEQ ID NO. 8, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO. 8. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 8. In some embodiments, the polypeptide derived from escherichia coli, or fragment thereof, comprises the pilin domain of escherichia coli FimH. Other suitable polypeptides derived from E.coli FimH and fragments thereof include FimH pilin domain variants having varying degrees of identity to SEQ ID NO:24, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 24. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 24. Other suitable polypeptides derived from e.coli FimH and fragments thereof include FimH lectin domain variants having varying degrees of identity to SEQ ID No. 26, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 26. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID No. 26.
In some embodiments, the composition comprises a polypeptide having at least n consecutive amino acids from any one of the following sequences: 3, 7, 8, 24 and 26, wherein n is 7 or greater (e.g., 8,10,12,14,16,18,20 or greater). Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of any one of the following sequences: 3, 7, 8, 24 and 26.
The position and length of the lectin domain of e.coli FimH or a homologue or variant thereof can be predicted based on a pairwise alignment of its sequence with any of the following sequences (e.g. by aligning the amino acid sequence of FimH with SEQ ID NO:1 and identifying the sequence aligned with residues 22-179 of SEQ ID NO: 1): 3, 7, 8, 24 and 26.
D. Wild type N-terminal signal sequence
In some embodiments, the N-terminal wild-type signal sequence of full-length FimH is cleaved in the host cell to produce the mature FimH polypeptide. Thus, FimH expressed by the host cell may lack the N-terminal signal sequence. In a preferred embodiment, the polypeptide derived from E.coli or a fragment thereof may be encoded by a nucleotide sequence lacking the coding sequence of the wild-type N-terminal signal sequence.
In some embodiments, the E.coli-derived polypeptide or fragment thereof comprises a FimH-FimC complex-forming fragment of FimH, an N-terminal signal sequence (such as residues 1-21 of SEQ ID NO: 1), or a combination thereof. A complex-forming fragment of FimH may be any portion or part of a FimH protein that retains the ability to form a complex with FimC.
In some embodiments, a polypeptide derived from e.coli, or fragment thereof, can lack 1 to 21 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, or lack 1-21, 1-20, 1-15, 1-10, 2-20, 2-15, 2-10, 5-20, 5-15, or 5-10 residues) at the N-terminus and/or C-terminus of the full-length FimH polypeptide, which can include a signal sequence, a lectin domain, and a pilin domain.
Nucleic acids
In one aspect, nucleic acids encoding polypeptides derived from E.coli, or fragments thereof, are disclosed. One or more nucleic acid constructs encoding polypeptides derived from E.coli, or fragments thereof, may be used for genomic integration and subsequent expression of polypeptides derived from E.coli, or fragments thereof. For example, a single nucleic acid construct encoding a polypeptide derived from E.coli or a fragment thereof can be introduced into a host cell. Alternatively, the coding sequence for the E.coli-derived polypeptide or fragment thereof may be carried by two or more nucleic acid constructs, which are then introduced into the host cell simultaneously or sequentially.
For example, in one exemplary embodiment, a single nucleic acid construct encodes the lectin domain and pilin domain of e.coli FimH. In another exemplary embodiment, one nucleic acid construct encodes the lectin domain and a second nucleic acid construct encodes the pilin domain of e.coli FimH. In some embodiments, genomic integration is achieved.
The nucleic acid construct may comprise genomic DNA comprising one or more introns or cdnas. When introns are present, expression of some genes is more efficient. In some embodiments, the nucleic acid sequence is suitable for expressing an exogenous polypeptide in said mammalian cell.
In some embodiments, the nucleic acid encoding the polypeptide or fragment thereof is codon optimized to increase expression levels in any particular cell.
In some embodiments, the nucleic acid construct comprises a signal sequence encoding a peptide that directs the secretion of a polypeptide derived from e. In some embodiments, the nucleic acid comprises a native signal sequence of a polypeptide derived from escherichia coli FimH. In some embodiments, wherein the polypeptide derived from E.coli or fragment thereof comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence may be codon optimized to increase the expression level of the protein in the host cell.
In some embodiments, the signal sequence is any one of the following lengths: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids in length. In some embodiments, the signal sequence is 20 amino acids in length. In some embodiments, the signal sequence is 21 amino acids in length.
In some embodiments, when the polypeptide or fragment thereof includes a signal sequence, the endogenous signal sequence naturally associated with the polypeptide can be replaced with a signal sequence unrelated to the wild-type polypeptide to increase the level of expression of the polypeptide or fragment thereof in the cultured cells. Thus, in some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide derived from E.coli, or a fragment thereof. In some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide derived from escherichia coli FimH. In some embodiments, the polypeptide derived from E.coli or a fragment thereof may be expressed together with a heterologous peptide, preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide derived from E.coli or a fragment thereof. For example, a polypeptide derived from E.coli FimH or a fragment thereof may be expressed together with a heterologous peptide (e.g., an IgK signal sequence), preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature E.coli FimH protein. In a preferred embodiment, the specific cleavage site at the N-terminus of the mature E.coli FimH protein occurs just before the initial phenylalanine residue of the mature E.coli FimH protein. The heterologous sequence of choice is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
In a preferred embodiment, the signal sequence is an IgK signal sequence. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO 18. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO 19. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO. 22. In a preferred embodiment, the signal sequence is a mouse IgK signal sequence.
Suitable mammalian expression vectors for the production of E.coli-derived polypeptides or fragments thereof are known in the art and may be commercially available, such as InvitrogenTMThe pSecTag2 expression vector of (1). An exemplary mouse Ig Kappa signal peptide sequence includes sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO: 54). In some embodiments, the vector comprises a pBudCE4.1 mammalian expression vector from Thermo Fisher. Other exemplary and suitable vectors include pcDNATM3.1 mammalian expression vector (Thermo Fisher).
In some embodiments, the signal sequence does not include a hemagglutinin signal sequence.
In some embodiments, the nucleic acid comprises a native signal sequence derived from a polypeptide of e. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence comprises a hemagglutinin signal sequence.
In one aspect, disclosed herein is a vector comprising a coding sequence for a polypeptide derived from escherichia coli, or a fragment thereof. Exemplary vectors include plasmids capable of autonomous replication or replication in mammalian cells. Typical expression vectors contain suitable promoters, enhancers, and terminators, which can be used to regulate the expression of one or more coding sequences in the expression construct. The vector may also comprise a selectable marker to provide a phenotypic trait (such as conferring resistance to an antibiotic such as ampicillin or neomycin) for selection of transformed host cells.
Suitable promoters are known in the art. Exemplary promoters include, for example, the CMV promoter, the adenovirus promoter, the EF1 a promoter, the GAPDH metallothionein promoter, the SV-40 early promoter, the SV-40 late promoter, the murine mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, and the like. Promoters may be constitutive or inducible. One or more vectors (e.g., one vector encoding all of its subunits or domains or fragments, or multiple vectors encoding together its subunits or domains or fragments) may be used.
Internal Ribosome Entry Sites (IRES) and 2A peptide sequences can also be used. IRES and 2A peptides provide an alternative method for the co-expression of multiple sequences. An IRES is a nucleotide sequence that allows translation to be initiated in the middle of a messenger rna (mrna) sequence as part of a larger process of protein synthesis. Generally, in eukaryotes, translation can only start from the 5' end of the mRNA molecule. IRES elements allow expression of multiple genes in one transcript. IRES-based polycistronic vectors express multiple proteins from a single transcript, which can reduce escape of non-expressing clones from selection. The 2A peptide allows translation of multiple proteins in a single open reading frame into a polyprotein (polyprotein) which is subsequently cleaved into individual proteins by the ribosome skipping mechanism. The 2A peptide can provide more balanced expression of multiple protein products. Exemplary IRES sequences include, for example, EV71 IRES, EMCV IRES, HCV IRES. For genomic integration, integration may be site-specific or random. Site-specific recombination can be achieved by introducing one or more homologous sequences into the nucleic acid constructs described herein. Such homologous sequences substantially match the endogenous sequence of a particular target site in the host genome. Alternatively, random integration may be used. Sometimes, the expression level of a protein may vary depending on the integration site. Thus, it may be desirable to select multiple clones based on the level of recombinant protein expression in order to identify clones that achieve the desired level of expression.
Exemplary nucleic acid constructs are further described in the figures, such as any of figures 2A-2T.
In one aspect, the nucleic acid sequence encodes an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identity to any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, 24, 26, 27, 28 and 29.
Host cell
In one aspect, the invention relates to a cell wherein a sequence encoding a polypeptide derived from E.coli or a fragment thereof is expressed in a mammalian host cell. In one embodiment, the polypeptide derived from E.coli or a fragment thereof is transiently expressed in a host cell. In another embodiment, the E.coli-derived polypeptide or fragment thereof is stably integrated into the genome of the host cell and, when cultured under suitable conditions, expresses the E.coli-derived polypeptide or fragment thereof. In preferred embodiments, the polynucleotide sequence is expressed with high efficiency and genomic stability.
Suitable mammalian host cells are known in the art. Preferably, the host cell is suitable for the production of proteins on an industrial production scale. Exemplary mammalian host cells include any of the following and derivatives thereof: chinese Hamster Ovary (CHO) cells, COS cells (monkey kidney (african green monkey) derived cell line), Vero cells, Hela cells, Baby Hamster Kidney (BHK) cells, Human Embryonic Kidney (HEK) cells, NSO cells (murine myeloma cell line) and C127 cells (non-tumorigenic mouse cell line). Other exemplary mammalian host cells include mouse Sertoli (TM4), buffalo rat liver (BRL 3A), Mouse Mammary Tumor (MMT), rat Hepatoma (HTC), mouse myeloma (NSO), murine hybridoma (Sp2/0), mouse thymoma (EL4), Chinese Hamster Ovary (CHO) and CHO cell derivatives, murine embryos (NIH/3T3, 3T3 Li), rat myocardium (H9C2), mouse myoblasts (C2C12), and mouse kidney (miMCD-3). Other examples of mammalian cell lines include NS0/1, Sp2/0, Hep G2, PER. C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL3A, W138, MMT 060562, TR1, MRC5, and FS 4.
According to the present invention, any cell susceptible to cell culture can be utilized according to the present invention. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used according to the present invention include BALB/c mouse myeloma cell line (NSO/l, ECACC No. 85110503); human retinoblasts (per. c6, CruCell, Leiden, The Netherlands); SV40 transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, j.gen virol.,36:59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216,1980); mouse support cells (TM4, Mather, biol. reprod.,23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.,383:44-68,1982); MRC5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). In some preferred embodiments, the cell is a CHO cell. In some preferred embodiments, the cell is a GS cell.
In addition, any number of commercially and non-commercially available hybridoma cell lines may be used in accordance with the present invention. As used herein, the term "hybridoma" refers to a cell or progeny of a cell resulting from the fusion of an immortalized cell and an antibody-producing cell. Such resulting hybridomas are antibody-producing immortalized cells. The individual cells used to produce the hybridomas may be from any mammalian source, including but not limited to rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments, the hybridoma is a trioma cell line that is produced when progeny of a heterohybrid myeloma fusion are subsequently fused with plasma cells, which is the product of a fusion of a human cell with a murine myeloma cell line. In some embodiments, the hybridoma is any immortalized hybrid cell line that produces antibodies, e.g., a quadroma (see, e.g., Milstein et al, Nature,537:3053,1983). One skilled in the art will appreciate that hybridoma cell lines may have different nutritional requirements and/or may require different culture conditions for optimal growth, and that the conditions will be able to be varied as desired.
In some embodiments, the cell comprises a first gene of interest, wherein the first gene of interest is chromosomally integrated. In some embodiments, the first gene of interest comprises a reporter gene, a selection gene, a gene of interest (e.g., encoding a polypeptide derived from e.coli, or a fragment thereof), an accessory gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a protein that is difficult to express (DtE).
In some embodiments, the first gene of interest is located between two different Recombination Target Sites (RTS) in a site-specific integration (SSI) mammalian cell, wherein the two RTS are chromosomally integrated within the NL1 locus or the NL2 locus. See, e.g., U.S. patent application publication No. 20200002727 for a description of the NL1 locus, the NL2 locus, the NL3 locus, the NL4 locus, the NL5 locus, and the NL6 locus. In some embodiments, the first gene of interest is located within the NL1 locus. In some embodiments, the cell comprises a second gene of interest, wherein the second gene of interest is chromosomally integrated. In some embodiments, the second gene of interest comprises a reporter gene, a selection gene, a therapeutic gene of interest (such as a polypeptide derived from e.coli, or a fragment thereof), an accessory gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding DtE protein. In some embodiments, the second gene of interest is located between two RTS. In some embodiments, the second gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the first gene of interest is located within the NL1 locus and the second gene of interest is located within the NL2 locus. In some embodiments, the cell comprises a third gene of interest, wherein the third gene of interest is chromosomally integrated. In some embodiments, the third gene of interest comprises a reporter gene, a selection gene, a therapeutic gene of interest (such as a polypeptide derived from e.coli, or a fragment thereof), an accessory gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding DtE protein. In some embodiments, the third gene of interest is located between two RTS. In some embodiments, the third gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the third gene of interest is located within a locus other than the NL1 locus and the NL2 locus. In some embodiments, the first gene of interest, the second gene of interest, and the third gene of interest are located within three separate loci. In some embodiments, at least one of the first, second, and third genes of interest is within the NL1 locus and at least one of the first, second, and third genes of interest is within the NL2 locus. In some embodiments, the cell comprises a site-specific recombinase gene. In some embodiments, the site-specific recombinase gene is chromosomally integrated.
In some embodiments, the present disclosure provides a mammalian cell comprising at least four different RTS, wherein the cell comprises (a) chromosomal integration of at least two different RTS at an NL1 locus or an NL2 locus; (b) a first gene of interest integrated between the at least two RTS of (a), wherein the first gene of interest comprises a reporter gene, a gene encoding DtE protein, an accessory gene, or a combination thereof; (c) and integrating a second gene of interest into a second chromosomal locus different from the locus of (a), wherein the second gene of interest comprises a reporter gene, a gene encoding an DtE protein (such as a polypeptide derived from e.coli, or a fragment thereof), an accessory gene, or a combination thereof. In some embodiments, the present disclosure provides a mammalian cell comprising at least four different RTS, wherein the cell comprises (a) chromosomal integration of at least two different RTS at the Fer1L4 locus; (b) at least two different RTS are chromosomally integrated within the NL1 locus or the NL2 locus; (c) a first gene of interest is chromosomally integrated within the Fer1L4 locus, wherein the first gene of interest comprises a reporter gene, a gene encoding a DtE protein, an accessory gene, or a combination thereof; and (d) chromosomal integration of a second gene of interest in the NL1 locus or the NL2 locus of (b), wherein the second gene of interest comprises a reporter gene, a gene encoding a DtE protein (e.g., a polypeptide derived from e.coli, or a fragment thereof), a helper gene, or a combination thereof.
In some embodiments, the present disclosure provides a mammalian cell comprising at least six different RTS, wherein the cell comprises (a) chromosomal integration of at least two different RTS and a first gene of interest within the Fer1L4 locus; (b) at least two different RTS and a second gene of interest are chromosomally integrated within the NL1 locus; and (c) at least two different RTS and a third gene of interest are chromosomally integrated within the NL2 locus.
As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linkage of nucleic acid sequences in such a way that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a way that a functional protein is produced. In some embodiments, the gene of interest is operably linked to a promoter, wherein the gene of interest is chromosomally integrated into the host cell. In some embodiments, the gene of interest is operably linked to a heterologous promoter; wherein the gene of interest is chromosomally integrated into the host cell. In some embodiments, the helper gene is operably linked to a promoter, wherein the helper gene is chromosomally integrated into the host cell genome. In some embodiments, the helper gene is operably linked to a heterologous promoter; wherein the helper gene is chromosomally integrated into the host cell genome. In some embodiments, the gene encoding DtE protein is operably linked to a promoter, wherein the gene encoding DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the gene encoding DtE protein is operably linked to a heterologous promoter, wherein the gene encoding DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a promoter, wherein the recombinase gene is chromosomally integrated into the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, wherein the recombinase gene is not integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally integrated into the host cell genome.
As used herein, the term "chromosomally integrated" or "chromosomal integration" refers to a nucleic acid sequence that is stably incorporated into the chromosome of a host cell (e.g., a mammalian cell), i.e., chromosomally integrated into the genomic dna (gdna) of the host cell (e.g., a mammalian cell). In some embodiments, the chromosomally integrated nucleic acid sequence is stable. In some embodiments, the chromosomally integrated nucleic acid sequence is not located on a plasmid or vector. In some embodiments, the chromosomally integrated nucleic acid sequence is not excised. In some embodiments, chromosomal integration is mediated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) gene editing systems (CRISPR/Cas).
In some embodiments, the host cell is suitable for growth in suspension culture. Suspension competent host cells are typically monodisperse or grow in loose aggregates without significant aggregation. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that are rendered suspension-competent by modification or adaptation of attachment-dependent cells (e.g., epithelial cells, fibroblasts).
In some embodiments, the expression level or activity of the e.coli-derived polypeptide or fragment thereof is increased by at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 90 fold, at least 100 fold as compared to the expression of the e.coli-derived polypeptide or fragment thereof in a bacterial cell (e.g., e.coli host cell).
The host cells described herein are suitable for large scale culture. For example, the cell culture may be 10L,30L,50L,100L,150L,200L,300L,500L,1000L,2000L,3000L,4000L,5000L,10,000L or greater. In some embodiments, the cell culture scale may range from 10L to 5000L,10L to 10,000L,10L, to 20,000L,10I, to 50,000L,40I, to 50,000L,100L to 50,000L,500L to 50,000L,1000L to 50,000L,2000L to 50,000L,3000I, to 50,000L,4000L to 50,000L,4500L to 50,000L,1000L to 10,000L,1000L to 20,000L,1000L to 25,000L,1000L to 30,000L,15L to 2000L,40L to 1000L,100L to 500L,200L to 400L, or any integer therebetween. Media components for cell culture are known in the art and may include, for example, buffers, amino acid components, vitamin components, salt components, mineral components, serum components, carbon source components, lipid components, nucleic acid components, hormone components, trace element components, ammonia components, cofactor components, indicator components, small molecule components, hydrolysate components, and enzyme modulator components.
As used herein, the terms "medium", "cell culture medium" and "culture medium" refer to a solution containing nutrients for a mammalian cell in vegetative growth. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and/or survival. Such solutions may also contain supplemental ingredients that enhance growth and/or survival above a minimum rate, including but not limited to hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), inorganic compounds present at high final concentrations (e.g., iron), amino acids, lipids, and/or glucose or other energy sources. In some embodiments, the culture medium is advantageously formulated at a pH and salt concentration that is optimal for cell survival and proliferation. In some embodiments, the medium is a feed medium added after the start of cell culture.
In some embodiments, cells can be grown in one of a variety of chemically defined media, wherein the composition of the media is known and controlled. In some embodiments, cells may be grown in complex media, where not all components of the media are known and/or controllable. Chemically defined growth media for mammalian cell culture have been widely developed and published over the past decades. All components of a defined medium are well characterized and therefore a defined medium does not contain complex additives such as serum or hydrolysates. Early media preparations were developed to allow cells to grow and maintain viability with little or no concern about protein production. Recently, media formulations have been developed with the clear aim of supporting high yield recombinant protein producing cell cultures. Such a medium is preferably used in the method of the present invention. Such media typically contain a large amount of nutrients, particularly amino acids, to support the growth and/or maintenance of cells at high density. These media can be modified by the skilled person if necessary for use in the method of the invention. For example, the skilled person can reduce the amount of phenylalanine, tyrosine, tryptophan and/or methionine in these media in order to use them as a basal medium or a feed medium in the methods disclosed herein.
Not all components of the complex medium are well characterized and therefore the complex medium may contain additives such as simple and/or complex carbon sources, simple and/or complex nitrogen sources, and serum, among others. In some embodiments, a complex medium suitable for use in the present invention comprises additives such as hydrolysates, in addition to other components of the defined media described herein. In some embodiments, a well-defined medium typically includes about fifty chemical entities at known concentrations in water. Most of these also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin, or BSA, but other media do not require protein components and are therefore referred to as protein-free, well-defined media. Typical chemical components of the culture medium are divided into five major groups: amino acids, vitamins, inorganic salts, trace elements and miscellaneous items that are difficult to classify simply.
The cell culture medium may optionally be supplemented with supplementary components. As used herein, the term "supplemental component" refers to a component that enhances growth and/or survival at a rate above a minimum, including, but not limited to, hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), amino acids, lipids, and/or glucose or other energy sources. In some embodiments, supplemental components may be added to the initial cell culture. In some embodiments, supplemental ingredients may be added after the cell culture has begun. Generally, trace elements refer to various inorganic salts contained at micromolar or lower levels. For example, zinc, selenium, copper, and the like are generally contained as trace elements. In some embodiments, iron (ferrous or ferric salts) may be included in the initial cell culture medium as a trace element at micromolar concentrations. Manganese is also often present as a divalent cation MnCl 2Or MnSO4) Included in the trace elements in a concentration range of nanomolar to micromolar. Many of the less common trace elements are typically added in nanomolar concentrations.
In some embodiments, the medium used in the methods of the invention is suitable for supporting high cell densities in cell culture (e.g., 1x 10)6Individual cells/mL, 5X106Individual cells/mL, 1X107Individual cells/mL, 5X107Individual cells/mL, 1X108Individual cell/mL or 5X108Individual cells/mL) of culture medium. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cell fed-batch culture.
In some embodiments, the cell culture medium comprises phenylalanine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises serine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises threonine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tyrosine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tryptophan at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and tryptophan at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, and tryptophan at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tryptophan, and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine, tryptophan, and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, and methionine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2mM, less than 1mM, between 0.1 and 2mM, between 0.1 and 1mM, between 0.5 and 1.5mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, preferably 2 mM. In some embodiments, the cell culture medium further comprises at least 5 of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, preferably 2 mM. In some embodiments, the cell culture medium further comprises glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, preferably 2 mM. In some embodiments, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, or preferably 2 mM. In some embodiments, the cell culture medium further comprises at least 5 of valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, preferably 2 mM. In some embodiments, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration greater than 2mM,3mM,4mM,5mM,10mM,15mM, preferably 2 mM. In some embodiments, the cell culture medium comprises serine at a concentration of greater than 3mM,5mM,7mM,10mM,15mM or 20mM, preferably 10 mM. In some embodiments, the cell culture medium comprises valine at a concentration of greater than 3mM,5mM,7mM,10mM,15mM, or 20mM, preferably 10 mM. In some embodiments, the cell culture medium comprises cysteine at a concentration of greater than 3mM,5mM,7mM,10mM,15mM or 20mM, preferably 10 mM. In some embodiments, the cell culture medium comprises isoleucine at a concentration greater than 3mM,5mM,7mM,10mM,15mM, or 20mM, preferably 10 mM. In some embodiments, the cell culture medium comprises leucine in a concentration of more than 3mM,5mM,7mM,10mM,15mM or 20mM, preferably 10 mM. In some embodiments, the cell culture medium described above is used in the methods disclosed herein. In some embodiments, the cell culture medium described above is used as a basal medium in the methods disclosed herein. In some embodiments, the above cell culture medium is used as a feed medium in the methods disclosed herein.
Production method
In one aspect, the invention includes a method of producing a polypeptide derived from E.coli, or a fragment thereof. The method comprises culturing mammalian cells under suitable conditions to express a polypeptide derived from E.coli, or a fragment thereof. The method may further comprise harvesting the polypeptide derived from E.coli or a fragment thereof from the culture. The method may further comprise purifying the polypeptide derived from E.coli or a fragment thereof.
In some embodiments, the methods produce the polypeptide or fragment thereof in a yield of 0.1g/L to 0.5 g/L.
In some embodiments, the cells may be grown in batch or fed-batch culture, wherein the culture is terminated after the polypeptide is fully expressed, after which the expressed polypeptide is harvested and optionally purified. In some embodiments, cells may be grown in perfusion cultures, wherein the culture is not terminated and new nutrients and other components are periodically or continuously added to the culture during which the expressed polypeptide is periodically or continuously harvested.
In some embodiments, cells can be grown in small scale reaction vessels ranging in volume from a few milliliters to a few. In some embodiments, cells may be grown in a large scale commercial bioreactor having a volume ranging from about 1 liter to 10 liters, 100 liters, 250 liters, 500 liters, 1000 liters, 2500 liters, 5000 liters, 8000 liters, 10000 liters, 12000 liters or more, or any volume in between.
The temperature of the cell culture will be selected based primarily on the temperature range at which the cell culture remains viable, the temperature range at which high levels of polypeptide are produced, the temperature range at which minimal metabolic waste is produced or accumulated, and/or any combination of these or other factors deemed important by the practitioner. As a non-limiting example, CHO cells grow well and produce high levels of protein or polypeptide at about 37 ℃. In general, most mammalian cells grow well in the range of about 25 ℃ to 42 ℃ and/or can produce high levels of protein or polypeptide, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells grow well in the range of about 35 ℃ to 40 ℃ and/or can produce high levels of protein or polypeptide. In certain embodiments, the cell culture is grown at a temperature of 20 ℃,21 ℃,22 ℃,23 ℃,24 ℃,25 ℃,26 ℃,27 ℃,28 ℃,29 ℃,30 ℃,31 ℃,32 ℃,33 ℃,34 ℃,35 ℃,36 ℃,37 ℃,38 ℃,39 ℃,40 ℃,41 ℃,42 ℃,43 ℃,44 ℃, or 45 ℃ at one or more times during the cell culture process.
As used herein, the terms "culture" and "cell culture" refer to a population of cells suspended in a culture medium under conditions suitable for survival and/or growth of the population of cells. It will be apparent to one of ordinary skill in the art that in some embodiments, these terms as used herein refer to a combination comprising a population of cells and a medium in which the population of cells is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.
The invention may be used with any cell culture method suitable for the desired process, such as the production of recombinant proteins (e.g. antibodies). As a non-limiting example, cells can be grown in batch or fed-batch culture, wherein the culture is terminated after sufficient expression of the recombinant protein (e.g., antibody), after which the expressed protein (e.g., antibody) is harvested. Alternatively, as another non-limiting example, the cells may be grown in a fed-batch mode, wherein the culture is not terminated and new nutrients and other ingredients are periodically or continuously added to the culture during which the expressed recombinant protein (e.g., antibody) is periodically or continuously harvested. Other suitable methods (e.g., centrifuge tube culture) are known in the art and may be used in the practice of the present invention.
In some embodiments, the cell culture suitable for use in the present invention is a fed-batch culture. As used herein, the term "fed-batch culture" is a method of culturing cells in which additional components are provided to the culture at one or more times after the start of the culturing process. Such provided components typically comprise nutrients for cells that have been depleted during the culturing process. Fed-batch culture is usually stopped at some point and the cells and/or components in the culture medium are harvested and optionally purified. In some embodiments, the fed-batch culture comprises a basal medium supplemented with a feed medium.
The cells may be grown in any convenient volume chosen by the practitioner. For example, cells can be grown in small scale reaction vessels with volumes ranging from milliliters to liters. Alternatively, cells may be grown in large scale commercial bioreactors having volumes ranging from about at least 1 liter to 10 liters, 50 liters, 100 liters, 250 liters, 500 liters, 1000 liters, 2500 liters, 5000 liters, 8000 liters, 10,000 liters, 12,000,15000 liters, 20000 liters or 25000 liters or more, or any volume therebetween.
The temperature of the cell culture will be selected primarily based on the temperature range at which the cell culture remains viable and the temperature range at which high levels of the desired product (e.g., recombinant protein) are produced. In general, most mammalian cells grow well and produce the desired product (e.g., recombinant protein) in the range of about 25 ℃ to 42 ℃, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells grow well in the range of about 35 ℃ to 40 ℃ and are capable of producing a desired product (e.g., a recombinant protein or antibody). In certain embodiments, the cell culture is cultured at a temperature of 20 ℃,21 ℃,22 ℃,23 ℃,24 ℃,25 ℃,26 ℃,27 ℃,28 ℃,29 ℃,30 ℃,31 ℃,32 ℃,33 ℃,34 ℃,35 ℃,36 ℃,37 ℃,38 ℃,39 ℃,40 ℃,41 ℃,42 ℃,43 ℃,44 ℃, or 45 ℃ at one or more times during the cell culture process. One of ordinary skill in the art will be able to select one or more suitable temperatures for growing cells depending on the particular needs of the cells and the particular production requirements of the practitioner. The cells may be grown for any length of time depending on the needs of the practitioner and the requirements of the cell itself. In some embodiments, the cells are grown at 37 ℃. In some embodiments, the cells are grown at 36.5 ℃.
In some embodiments, cells may be grown for longer or shorter periods of time during the initial growth phase (or growth phase), depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown for a period of time sufficient to achieve a predetermined cell density. In some embodiments, the cells are grown for a period of time sufficient to reach a cell density that is a given percentage of the maximum cell density that the cells will eventually reach if allowed to grow undisturbed. For example, the cells may be grown for a period of time sufficient to achieve a desired viable cell density of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the maximum cell density. In some embodiments, the cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In some embodiments, the cells are grown until the cell density does not increase more than 5% per day of culture.
In some embodiments, the cells are grown for a defined period of time. For example, depending on the initial concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or more, preferably 4 to 10 days. In some cases, the cells may be grown for one month or more. The practitioner of the invention will be able to select the duration of the initial growth phase according to the protein production requirements and the needs of the cell itself.
During the initial culture period, the cell culture may be agitated or shaken to increase oxygenation of the cells and dispersion of nutrients into the cells. In light of the present disclosure, one of ordinary skill in the art will appreciate that it may be beneficial to control or adjust certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, and the like.
At the end of the initial growth phase, at least one culture condition may be shifted such that a second set of culture conditions can be applied and a metabolic shift occurs in the culture. Metabolic conversion can be achieved by, for example, altering the temperature, pH, osmolality, or chemical inducer levels of the cell culture. In one non-limiting embodiment, the culture conditions are shifted by shifting the temperature of the culture. However, as is known in the art, the shift temperature is not the only mechanism to achieve an appropriate metabolic shift. For example, such metabolic conversion may also be achieved by shifting other culture conditions, including but not limited to pH, osmolality, and sodium butyrate levels. The timing of the culture transition will be determined by the practitioner of the invention according to the protein production requirements or the needs of the cell itself.
When changing the temperature of the culture, the temperature change may be relatively gradual. For example, it may take hours or days to complete a temperature shift. Alternatively, the temperature transition may be relatively abrupt. For example, the temperature shift may be completed in less than a few hours. Given appropriate production and control equipment, such as standard equipment in commercial large-scale production of polypeptides or proteins, temperature shifts can be completed even in less than an hour.
In some embodiments, once the conditions of the cell culture are altered as described above, the cell culture may be maintained for a subsequent production phase under a second set of culture conditions that contribute to the survival and viability of the cell culture and are suitable for expressing the desired polypeptide or protein at commercially appropriate levels.
As noted above, the culture can be shifted by changing one or more of a number of culture conditions, including but not limited to temperature, pH, osmolality, and sodium butyrate levels. In some embodiments, the temperature of the culture is varied. According to this embodiment, the culture is maintained at a temperature or temperature range below that of the initial growth phase during the subsequent production phase. As described above, multiple discrete temperature shifts may be employed to increase cell density or viability, or to increase expression of recombinant proteins.
In some embodiments, the cells may be maintained in a subsequent production phase until a desired cell density or production titer is reached. In another embodiment of the invention, the cells are grown for a defined period of time during the subsequent production phase. For example, cells may be grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or more depending on the concentration of the cell culture at the beginning of the subsequent growth phase, the temperature at which the cells are grown, and the intrinsic growth rate of the cells. In some cases, the cells may be grown for one month or more. The practitioner of the invention will be able to select the duration of the subsequent production phase according to the polypeptide or protein production requirements and the needs of the cell itself.
During the subsequent production phase, the cell culture may be agitated or shaken to increase oxygenation and diffusion of nutrients into the cells. In accordance with the present invention, one of ordinary skill in the art will appreciate that it may be beneficial to control or adjust certain internal conditions of the bioreactor during subsequent growth phases, including but not limited to pH, temperature, oxygenation, and the like.
In some embodiments, the cell expresses a recombinant protein, and the cell culture methods of the invention comprise a growth phase and a production phase.
In some embodiments, step (ii) of any of the methods disclosed herein is applied during the entire cell culture process. In some embodiments, step (ii) of any of the methods disclosed herein is applied during a portion of a cell culture process. In some embodiments, step (ii) is applied until a predetermined viable cell density is obtained.
In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during a portion of the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during the growth phase and the production phase.
In step (ii) of any of the methods disclosed herein, the term "maintaining" may refer to maintaining a concentration of amino acids or metabolites below C1 or C2 throughout the culture (until harvest) or during part of the culture (e.g., during the growth phase, part of the growth phase, or until a predetermined cell density is obtained).
In some embodiments of any of the above methods, the cell growth and/or productivity is increased as compared to a control culture that is the same except that it does not comprise step (ii).
In some embodiments of any of the above methods, the methods of the invention are methods for improving cell growth. In some embodiments, the methods of the invention are methods for improving cell growth in high density cell culture at high cell densities.
High cell density, as used herein, means greater than 1x106Individual cells/mL, 5X106Individual cells/mL, 1X107Individual cells/mL, 5X107Individual cells/mL, 1X108Individual cell/mL or 5X108Individual cells/mL, preferably greater than 1X107Individual cells/mL, more preferably greater than 5X107Cell density of individual cells/mL.
In some embodiments, the methods of the invention are methods for improving cell growth in cell culture, wherein the cell density is greater than 1x106Individual cells/mL, 5X106Individual cells/mL, 1X107Individual cells/mL, 5X107Individual cells/mL, 1X108Individual cell/mL or 5X108Individual cells/mL. In some embodiments, the methods of the invention are methods for improving cell growth in cell culture, wherein the maximum cell density is greater than 1x10 6Individual cells/mL, 5X106Individual cells/mL, 1X107Individual cells/mL, 5X107Individual cells/mL, 1X108Individual cell/mL or 5X108Individual cells/mL.
In some embodiments, cell growth is determined by Viable Cell Density (VCD), maximum viable cell density, or Integrated Viable Cell Count (IVCC). In some embodiments, cell growth is determined by the maximum viable cell density.
As used herein, the term "viable cell density" refers to the number of cells present in a given volume of culture medium. Viable cell density can be measured by any method known to the skilled person. Preferably, an automated cell counter such as BioProfile is used
Figure BDA0003622971360000481
Viable cell density was measured. As used herein, the term maximum cell density refers to the maximum cell density achieved during cell culture. As used herein, the term "cell viability" refers to the ability of a cell in culture to survive a given set of culture conditions or experimental variations. One of ordinary skill in the art will appreciate that the present invention encompasses one of many methods for determining cell viability. For example, cell viability may be determined using a dye (e.g., trypan blue) that does not cross the membrane of living cells, but may cross the ruptured membrane of dead or dying cells.
As used herein, the term "Integrated Viable Cell Count (IVCC)" refers to the area under the Viable Cell Density (VCD) curve. IVCC can be calculated using the following formula: IVCCt+1＝IVCCt+(VCDt+VCDt+1) (Δ t)/2, wherein Δ t is the time difference between the time point t and the time point t + 1. IVCC can be assumedt＝0Can be ignored. VCDtAnd VCDt+1Viable cell density was at t and t +1 time points.
As used herein, for example, the term "titer" refers to the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titers are usually expressed in grams of protein per liter of medium.
In some embodiments, cell growth is increased by at least 5%, 10%, 15%, 20%, or 25% as compared to a control culture. In some embodiments, cell growth is increased by at least 10% compared to a control culture. In some embodiments, cell growth is increased by at least 20% compared to a control culture.
In some embodiments, the productivity is determined by titer and/or volumetric productivity.
As used herein, for example, the term "titer" refers to the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titers are usually expressed in grams of protein per liter of medium.
In some embodiments, the productivity is determined by titer. In some embodiments, the productivity is increased by at least 5%, 10%, 15%, 20%, or 25% as compared to a control culture. In some embodiments, the productivity is increased by at least 10% compared to a control culture. In some embodiments, the productivity is increased by at least 20% compared to a control culture.
In some embodiments, the maximum cell density of the cell culture is greater than 1x106Individual cells/mL, 5X106Individual cells/mL, 1X107Individual cells/mL, 5X107Individual cells/mL, 1X108Individual cell/mL or 5X108Individual cells/mL. In some embodiments, the maximum cell density of the cell culture is greater than 5x106Individual cells/mL. In some embodiments, the maximum cell density of the cell culture is greater than 1x108Individual cells/mL.
V. purification
In some embodiments, the method for producing a polypeptide derived from escherichia coli or a fragment thereof comprises isolating and/or purifying the polypeptide derived from escherichia coli or a fragment thereof. In some embodiments, the expressed polypeptide or fragment thereof derived from E.coli is secreted into the culture medium, and thus cells and other solids can be removed by centrifugation and/or filtration.
The e.coli-derived polypeptides or fragments thereof produced according to the methods described herein can be harvested from the host cells and purified using any suitable method. Suitable methods for purifying a polypeptide or fragment thereof include precipitation and various types of chromatography (such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion), all of which are known in the art. Suitable purification schemes may include two or more of these or other suitable methods. In some embodiments, one or more polypeptides derived from e.coli or fragments thereof may comprise a "tag" that facilitates purification, such as an epitope tag or HIS tag, Strep tag. Such labelled polypeptides may conveniently be purified by chelate chromatography or affinity chromatography, for example from conditioned media. Optionally, the tag sequence may be cleaved after purification.
In some embodiments, the polypeptide derived from escherichia coli or fragment thereof can include a tag for affinity purification. Affinity purification tags are known in the art. Examples include e.g. His-tag (binding to metal ions), antibodies, Maltose Binding Protein (MBP) (binding to amylose), glutathione-S-transferase (GST) (binding to glutathione), FLAG-tag, Strep-tag (binding to streptavidin or derivatives thereof).
In a preferred embodiment, the polypeptide derived from E.coli or fragment thereof does not comprise a purification tag.
In some embodiments, the yield of a polypeptide derived from E.coli, or a fragment thereof, is at least about 1mg/L, at least about 2mg/L, at least about 3mg/L, at least about 4mg/L, at least about 5mg/L, at least about 6mg/L, at least about 7mg/L, at least about 8mg/L, at least about 9mg/L, at least about 10mg/L, at least about 11mg/L, at least about 12mg/L, at least about 13mg/L, at least about 14mg/L, at least about 15mg/L, at least about 16mg/L, at least about 17mg/L, at least about 18mg/L, at least about 19mg/L, at least about 20mg/L, at least about 25mg/L, at least about 30mg/L, at least about 35mg/L, at least about 40mg/L, at least about 45mg/L, at least about 50mg/L, at least about 55mg/L, at least about 60mg/L, at least about 65mg/L, at least about 70mg/L, at least about 75mg/L, at least about 80mg/L, at least about 85mg/L, at least about 90mg/L, at least about 95mg/L or, at least about 100 mg/L.
In some embodiments, the culture is on a scale of at least about 10 liters, e.g., a volume of at least about 10L, at least about 20L, at least about 30L, at least about 40L, at least about 50L, at least about 60L, at least about 70L, at least about 80L, at least about 90L, at least about 100L, at least about 150L, at least about 200L, at least about 250L, at least about 300L, at least about 400L, at least about 500L, at least about 600L, at least about 700L, at least about 800L, at least about 900L, at least about 1000L, at least about 2000L, at least about 3000L, at least about 4000L, at least about 5000L, at least about 6000L, at least about 10,000L, at least about 15,000L, at least about 20,000L, at least about 25,000L, at least about 30,000L, at least about 35,000L, at least about 40,000L, at least about 45,000L, at least about 50,000L, at least about 55,000L, at least about 65,000L, at least about 70,000L, at least about 75,000L, at least about 80,000L, at least about 85,000L, at least about 90,000L, at least about 95,000L, at least about 100,000L, and the like.
Compositions and formulations
In one aspect, the invention includes a composition comprising a polypeptide derived from E.coli, or a fragment thereof. In some embodiments, the composition elicits an immune response, including antibodies, that can confer immunity to a pathogenic species of escherichia coli.
In some embodiments, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof, as the sole antigen. In some embodiments, the composition does not comprise a conjugate.
In some embodiments, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof, and an additional antigen. In some embodiments, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof, and an additional escherichia coli antigen. In some embodiments, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof, and a glycoconjugate from escherichia coli.
In some embodiments, the polypeptide or fragment thereof is derived from escherichia coli FimH.
In some embodiments, the composition comprises a polypeptide derived from e.coli FimC or a fragment thereof.
In some embodiments, the composition comprises a polypeptide derived from escherichia coli FimH, or a fragment thereof; and a polypeptide derived from escherichia coli FimC or a fragment thereof.
In one aspect, the invention includes a composition comprising a polypeptide derived from escherichia coli FimH, or a fragment thereof; and a saccharide comprising a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and the strain of formula O5 (180/C)), formula O (for example, formula O: K; K and the strain of formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula I, formula O, formula I, formula O, formula I, formula O, formula I, formula O, formula I, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D1, formula O63, formula O64, formula O65 (for example formula O65 (strain 73-1)), formula O65, formula O685120, formula O65, formula O685126, formula O685120, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O685, formula O65, formula O685126, formula O685, formula O685126, formula O65, formula O685126, formula O685, formula O65, formula O685120, formula O65, formula O685120, formula O685126, formula O685, formula O685126, formula O65, formula O685, formula O685120, formula O685 formula O65, formula O685, formula O685120, formula O685, formula O685, formula O685, formula O65, formula O685 formula O103, formula O685 formula O65, formula O685 formula O103, formula O685, formula O103, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187, wherein n is an integer from 1 to 100.
In some embodiments, the composition comprises any one of the saccharides disclosed herein. In a preferred embodiment, the composition comprises any one of the conjugates disclosed herein.
In some embodiments, the composition comprises at least one glycoconjugate from escherichia coli serotype O25, preferably serotype O25 b. In one embodiment, the composition comprises at least one glycoconjugate from escherichia coli serotype O1, preferably serotype O1 a. In one embodiment, the composition comprises at least one glycoconjugate from e.coli serotype O2. In one embodiment, the composition comprises at least one glycoconjugate from e.coli serotype O6.
In one embodiment, the composition comprises at least one glycoconjugate selected from any one of the following escherichia coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least two glycoconjugates selected from any one of the following escherichia coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least three glycoconjugates selected from any one of the following escherichia coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises glycoconjugates from each of the following e.coli serotypes O25, O1, O2 and O6, preferably O25b, O1a, O2 and O6.
In a preferred embodiment, the glycoconjugates of any of the above compositions are separately conjugated to CRM197And (4) conjugation.
Thus, in some embodiments, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and an O-antigen from at least one E.coli serotype. In a preferred embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from more than one serotype of E.coli. For example, the composition may comprise O-antigens from two different e.coli serotypes (or "v", titers) to 12 different serotypes (12 v). In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 3 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 4 different E.coli serotypes. In one embodiment, the composition comprises O-antigens from 5 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 6 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 7 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 8 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 9 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 10 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 11 different E.coli serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 12 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 13 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 14 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 15 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 16 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 17 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 18 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 19 different serotypes. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 20 different serotypes.
Preferably, the number of e.coli saccharides may range from 1 serotype (or "v", titer) to 26 different serotypes (26 v). In one embodiment, there is one serotype. In one embodiment, there are 2 different serotypes. In one embodiment, there are 3 different serotypes. In one embodiment, there are 4 different serotypes. In one embodiment, there are 5 different serotypes. In one embodiment, there are 6 different serotypes. In one embodiment, there are 7 different serotypes. In one embodiment, there are 8 different serotypes. In one embodiment, there are 9 different serotypes. In one embodiment, there are 10 different serotypes. In one embodiment, there are 11 different serotypes. In one embodiment, there are 12 different serotypes. In one embodiment, there are 13 different serotypes. In one embodiment, there are 14 different serotypes. In one embodiment, there are 15 different serotypes. In one embodiment, there are 16 different serotypes. In one embodiment, there are 17 different serotypes. In one embodiment, there are 18 different serotypes. In one embodiment, there are 19 different serotypes. In one embodiment, there are 20 different serotypes. In one embodiment, there are 21 different serotypes. In one embodiment, there are 22 different serotypes. In one embodiment, there are 23 different serotypes. In one embodiment, there are 24 different serotypes. In one embodiment, there are 25 different serotypes. In one embodiment, there are 26 different serotypes. The saccharide is coupled to a carrier protein to form a glycoconjugate as described herein.
In one aspect, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and a glycoconjugate comprising an O-antigen from at least one e. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from more than one E.coli serotype, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 2 different E.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 3 different E.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 4 different E.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 5 different E.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 6 different e.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 7 different e.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 8 different E.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 9 different e.coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises an O-antigen derived from an e.coli polypeptide or fragment thereof; and 10 different E.coli serotypes, each of which is associated with a carrier protein. In one embodiment, the composition comprises an O-antigen derived from an e.coli polypeptide or fragment thereof; and 11 different E.coli serotypes, in which each O-antigen is associated with a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 12 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 13 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 14 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 15 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 16 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 17 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 18 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 19 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-antigens from 20 different serotypes, wherein each O-antigen is conjugated to a carrier protein.
In another aspect, the composition comprises O-polysaccharides from at least one E.coli serotype. In a preferred embodiment, the composition comprises O-polysaccharides from more than one E.coli serotype. For example, the composition can comprise O-polysaccharides from two different e.coli serotypes to 12 different e.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 3 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 4 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 5 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 6 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 7 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 8 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 9 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 10 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 11 different E.coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 12 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 13 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 14 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 15 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 16 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 17 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 18 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 19 different serotypes. In one embodiment, the composition comprises O-polysaccharides from 20 different serotypes.
In a preferred embodiment, the composition comprises an O-polysaccharide from at least one E.coli serotype, wherein the O-polysaccharide is conjugated to a carrier protein. In a preferred embodiment, the composition comprises O-polysaccharides from more than one E.coli serotype, wherein each O-polysaccharide is conjugated to a carrier protein. For example, the composition can comprise O-polysaccharides from two different e.coli serotypes to 12 different e.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 3 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 4 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 5 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 6 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 7 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 8 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 9 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 10 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 11 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 12 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 13 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 15 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 16 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 17 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 19 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein.
In a most preferred embodiment, the composition comprises an O-polysaccharide from at least one E.coli serotype, wherein the O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In a preferred embodiment, the composition comprises O-polysaccharides from more than one E.coli serotype, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core sugar. For example, the composition can include O-polysaccharides from two different e.coli serotypes to 12 different e.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide includes an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 3 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 4 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 5 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 6 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 7 different e.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 8 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 9 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides include O-antigen and core sugar. In one embodiment, the composition comprises O-polysaccharides from 10 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 11 different E.coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 12 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 13 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 15 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 16 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 17 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 19 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises O-polysaccharides from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In a preferred embodiment, the carrier protein is CRM197。
In another preferred embodiment, the composition comprises a polypeptide derived from E.coli or a fragment thereof; and CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O25a and a core sugar, wherein n is at least 40. In a preferred embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O25b and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O1a and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O2 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O6 and a core sugar, wherein n is at least 40.
In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O17 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O15 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM 197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises the formula O18A and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises the formula O75 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O4 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O16 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O13 and a core sugar, wherein n is at least 40. In another embodiment, the composition further comprises CRM197Conjugated O-polysaccharides, wherein O-is polySugars include those of the formula O7 and core sugars, where n is at least 40.
In another embodiment, the composition further comprises CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O8 and a core sugar, wherein n is at least 40. In another embodiment, the O-polysaccharide comprises the formula O8, wherein n is 1 to 20, preferably 2 to 5, more preferably 3. For example, formula O8 is shown in FIG. 10B. In another embodiment, the composition further comprises CRM 197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises the formula O9 and a core sugar, wherein n is at least 40. In another embodiment, the O-polysaccharide comprises the formula O9, wherein n is 1 to 20, preferably 4 to 8, more preferably 5. For example, formula O9 is shown in FIG. 10B. In another embodiment, the O-polysaccharide comprises the formula O9a, wherein n is 1 to 20, preferably 4 to 8, more preferably 5. For example, formula O9a is shown in fig. 10B.
In some embodiments, the O-polysaccharide comprises an O-polysaccharide selected from any one of the formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, wherein n is 1 to 20, preferably 4 to 8, more preferably 5. See, for example, fig. 10B.
As described above, the composition may comprise a polypeptide derived from escherichia coli, or a fragment thereof; and conjugated O-polysaccharide (antigen). In an exemplary embodiment, the composition comprises a polysaccharide comprising formula O25b, a polysaccharide comprising formula O1A, a polysaccharide comprising formula O2, and a polysaccharide comprising formula O6. More specifically, such as a composition comprising: (i) and CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O25b and a core sugar, wherein n is at least 40; (ii) and CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises formula O1a and a core sugar, wherein n is at least 40; (iii) and CRM 197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises the formula O2 and a core sugar, wherein n is at least 40; and (iv) with CRM197A conjugated O-polysaccharide, wherein the O-polysaccharide comprises the formula O6 and a core sugar, wherein n is at least 40.
In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and at least one O-polysaccharide derived from any E.coli serotype, wherein the serotype is not O25 a. For example, in one embodiment, the composition does not comprise a saccharide comprising the formula O25 a. Such compositions may comprise, for example, an O-polysaccharide comprising the formula O25b, an O-polysaccharide comprising the formula O1A, an O-polysaccharide comprising the formula O2, and an O-polysaccharide comprising the formula O6.
In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 2 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 3 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 4 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 5 different E.coli serotypes, wherein each O-polysaccharide is associated with CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 6 different E.coli serotypes, wherein each O-polysaccharide is associated with CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 7 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 8 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and from 9 different O-polysaccharides of E.coli serotypes, each of which is combined with CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 10 different E.coli serotypes, wherein each O-polysaccharide is associated with CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 11 different E.coli serotypes, wherein each O-polysaccharide is conjugated to CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 12 different serotypes, wherein each O-polysaccharide is conjugated to CRM197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 13 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 14 different serotypes, wherein each O-polysaccharide is conjugated to CRM 197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 15 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 16 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 17 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition packageContaining a polypeptide derived from Escherichia coli or a fragment thereof; and O-polysaccharides from 18 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 19 different serotypes, wherein each O-polysaccharide is conjugated to CRM 197And wherein the O-polysaccharide comprises an O-antigen and a core sugar. In one embodiment, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and O-polysaccharides from 20 different serotypes, wherein each O-polysaccharide is conjugated to CRM197Conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core sugar.
In one aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 15 ± 2. In one aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 17 ± 2. In one aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 55 ± 2. In another aspect, the invention relates to a composition comprising a polypeptide derived from Escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 51 ± 2. In one embodiment, the sugar further comprises an e.coli R1 core sugar moiety. In another embodiment, the sugar further comprises an e.coli K12 core sugar moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM 197. In one embodiment, the conjugate is prepared by single-ended conjugation. In one embodiment, the conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is interrupted by a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC)The daughter is conjugated to a carrier protein. Preferably, the composition further comprises a pharmaceutically acceptable diluent.
In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding to escherichia coli serotype O25B polysaccharide at a concentration of at least 0.2pg/ml, 0.3pg/ml, 0.35pg/ml, 0.4pg/ml, or 0.5pg/ml as determined by an ELISA assay. Thus, pre-and post-immune sera can be compared to OPA activity of the immunogenic compositions of the invention and their response to serotype O25B to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing escherichia coli serotype O25B as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing escherichia coli serotype O25B, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O25B compared to the pre-immune population. In one embodiment, the immunogenic composition elicits a titer against e.coli serotype O25B of at least 1:8 in at least 50% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against e.coli serotype O25B in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O25B compared to the pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titer against e.coli serotype O25B in human subjects compared to the pre-immune population.
On the one hand, the method comprises the following steps of,the present invention relates to compositions comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O1a, wherein n is 39 ± 2. In another aspect, the invention relates to a composition comprising a polypeptide derived from E.coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O1a, wherein n is 13 ± 2. In one embodiment, the saccharide further comprises an e.coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM197. In one embodiment, the conjugate is prepared by single-ended conjugation. In one embodiment, the conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.
In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding to escherichia coli serotype O1A polysaccharide at a concentration of at least 0.2pg/ml, 0.3pg/ml, 0.35pg/ml, 0.4pg/ml, or 0.5pg/ml as determined by an ELISA assay. Pre-and post-immune sera can be compared to OPA activity of the immunogenic compositions of the invention and their response to serotype O1A to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing escherichia coli serotype O1A as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing escherichia coli serotype O1A, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O1A compared to the pre-immune population. In one embodiment, the immunogenic composition elicits a titer against e.coli serotype O1A of at least 1:8 in at least 50% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against e.coli serotype O1A in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O1A compared to the pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titer against e.coli serotype O1A in human subjects compared to the pre-immune population.
In one aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 43 ± 2. In another aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 47 ± 2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 17 ± 2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 18 ± 2. In one embodiment, the sugar further comprises an e.coli R1 core sugar moiety. In another embodiment, the sugar further comprises an E.coli R4 core sugar moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM197. In one embodiment, the conjugate is prepared by single-ended conjugation. In one embodiment, the conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is substituted with (2- ((2-oxoethyl) thio) Para) ethyl) carbamate (eTEC) spacer is conjugated to a carrier protein. Preferably, the composition further comprises a pharmaceutically acceptable diluent.
In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding to escherichia coli serotype O2 polysaccharide at a concentration of at least 0.2pg/ml, 0.3pg/ml, 0.35pg/ml, 0.4pg/ml, or 0.5pg/ml as determined by an ELISA assay. Thus, pre-and post-immune sera can be compared to OPA activity of the immunogenic compositions of the invention and their response to serotype O2 to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing escherichia coli serotype O2 as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing escherichia coli serotype O2, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O2 compared to the pre-immune population. In one embodiment, the immunogenic composition elicits a titer against e.coli serotype O2 of at least 1:8 in at least 50% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against e.coli serotype O2 in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O2 compared to the pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titer against e.coli serotype O2 in human subjects compared to the pre-immune population.
In one aspect, the invention relates to a composition comprising a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 42 ± 2. In another aspect, the invention relates to a composition comprising a polypeptide derived from E.coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 50 ± 2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 17 ± 2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 18 ± 2. In one embodiment, the sugar further comprises an e.coli R1 core sugar moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM197. In one embodiment, the conjugate is prepared by single-ended conjugation. In one embodiment, the conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.
In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding to escherichia coli serotype O6 polysaccharide at a concentration of at least 0.2pg/ml, 0.3pg/ml, 0.35pg/ml, 0.4pg/ml, or 0.5pg/ml as determined by an ELISA assay. Thus, pre-and post-immune sera can be compared to OPA activity of the immunogenic compositions of the invention and their response to serotype O6 to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing escherichia coli serotype O6 as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing escherichia coli serotype O6, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O6 compared to the pre-immune population. In one embodiment, the immunogenic composition elicits a titer against e.coli serotype O6 of at least 1:8 in at least 50% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against e.coli serotype O6 in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (i.e., individuals with serum possessing a titer of at least 1:8 as determined by OPA in vitro) to e.coli serotype O6 compared to the pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titer against e.coli serotype O6 in human subjects compared to the pre-immune population.
In one aspect, the composition comprises a polypeptide derived from escherichia coli, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula 45, formula O, formula I52. Formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60 (for example formula O60 (73-1 strain)), formula O60, formula O120, formula O60, formula O124, formula O60, formula O103, formula O60, formula O68513, formula O60, formula O68513, formula O60, formula O68513, formula O60, formula O68513, formula O60, formula O685102, formula O68513, formula O60, formula O685102, formula O60, formula O685102, formula O60, formula O685102, formula O60, formula O685103, formula O60, formula O103, formula O685102, formula O60, formula O685103, formula O60, formula O685102, formula O60, formula O103, formula O60, formula O685103, formula O60, formula O103, formula O60, formula O103, formula O60, formula O103, formula O60, formula O103, formula O103, formula O60, formula O103 formula O60, formula O103 formula O formula 103 formula O formula 103 formula, Formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187, wherein n is an integer from 1 to 100. In one embodiment, the saccharide further comprises an e.coli R1 core saccharide moiety. In one embodiment, the sugar further comprises an e.coli R2 core sugar moiety. In one embodiment, the sugar further comprises an e.coli R3 core sugar moiety. In another embodiment, the sugar further comprises an E.coli R4 core sugar moiety. In one embodiment, the sugar further comprises an e.coli K12 core sugar moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM 197. In one embodiment, the conjugate is prepared by single-ended conjugation. In one embodiment, the conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is a sugar-free extractA per (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer is conjugated to the carrier protein. Preferably, the composition further comprises a pharmaceutically acceptable diluent. In one embodiment, the composition further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 additional conjugates each comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises a structure selected from any one of the formulas.
A. Saccharides and their use as anti-inflammatory agents
In one embodiment, the size of the saccharide is controlled by expressing (not necessarily overexpressing) a different Wzz protein (e.g., WzzB), thereby producing the saccharide.
As used herein, the term "saccharide" refers to a single saccharide moiety or monosaccharide unit, as well as a combination of two or more single saccharide moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides and polysaccharides. The sugar may be straight or branched.
In one embodiment, the sugar is produced in a recombinant gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant E.coli cell. In one embodiment, the saccharide is produced in a recombinant salmonella cell. Exemplary bacteria include Escherichia coli O25K5H1, Escherichia coli BD559, Escherichia coli GAR2831, Escherichia coli GAR865, Escherichia coli GAR868, Escherichia coli GAR869, Escherichia coli GAR872, Escherichia coli GAR878, Escherichia coli GAR896, Escherichia coli GAR1902, Escherichia coli O25a ETC NR-5, Escherichia coli O157H 7: K-, Salmonella enterica serotype typhimurium LT2 strain, Escherichia coli GAR2401, Salmonella serotype typhimurium CVD 1943, Salmonella serotype typhimurium CVD 1925, Salmonella serotype A paratyphoid CVD 1902 enterica, and Shigella flexneri CVD 1208. In one embodiment, the bacterium is not escherichia coli GAR 2401. This genetic approach to carbohydrate production allows for the efficient production of O-polysaccharides and O-antigen molecules as vaccine components.
As used herein, the term "wzz protein" refers to chain length blocksPolypeptide (chain length determining polypeptide), e.g. wzB, wzz SF、wzzST、fepE、wzzfepEWzzl and wzz 2. Exemplary wzz gene sequences have GenBank accession numbers AF011910 (for E4991/76), AF011911 (for F186), AF011912 (for M70/1-1), AF011913 (for 79/311), AF011914 (for Bi7509-41), AF011915 (for C664-1992), AF011916 (for C258-94), AF011917 (for C722-89), and AF011919 (for EDL 933). GenBank accession numbers of the G7 and Bi316-41wzz gene sequences are U39305 and U39306 respectively. Other GenBank accession numbers for the exemplary wzz gene sequence are NP _459581 (for salmonella enterica sub-species of the Salmonella enterica serotype LT2FepE strain), AIG66859 (for E.coli O157: H7 EDL933 FepE strain), NP _461024 (for salmonella enterica sub-species of the Salmonella enterica serotype LT2 WzzB strain). Np _416531 (for Escherichia coli K-12 sub-strain MG1655 WzzB), NP _415119 (for Escherichia coli K-12 sub-strain MG1655 FepE). In a preferred embodiment, the wzz family protein is wzzB, wzzSF、wzzST、fepE、wzzfepEAny of wzz1 and wzz2, most preferably wzzB, and more preferably fepE.
Exemplary wzzB sequences include those shown in SEQ ID Nos 30-34. Exemplary FepE sequences include those shown in SEQ ID Nos 35-39.
In some embodiments, modified sugars (modified compared to the corresponding wild-type sugars) can be produced by expressing (without over-expressing) wzz family proteins from gram-negative bacteria (e.g., fepE) in gram-negative bacteria and/or by turning off (i.e., inhibiting, deleting, removing) the second wzz gene (e.g., wzzB) to produce high molecular weight sugars (e.g., lipopolysaccharides) containing intermediate or long O-antigen chains. For example, a modified sugar can be produced by expressing (not necessarily overexpressing) wzz2 and turning off wzz 1. Alternatively, in the alternative, the modified sugar may be produced by expressing (not necessarily over-expressing) wzzfepE and turning off wzzB. In another embodiment, the modified sugar may be produced by expressing (not necessarily over expressing) wzzB but turning off wzzfepE. In another embodiment, the modified sugar may be produced by expression of fepE. Preferably, the wzz family protein is derived from a strain heterologous to the host cell.
In some embodiments, the saccharide is produced by expressing wzz a family protein, the wzz family protein having an amino acid sequence having at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of the following sequences: 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 SEQ ID NO. In one embodiment, the wzz family protein includes any one of the sequences selected from the group consisting of: 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 SEQ ID NO. Preferably, the wzz family protein has at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to: 30, 31, 32, 33, 34. In some embodiments, the saccharide is produced by expressing a protein having an amino acid sequence with at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the fepE protein.
In one aspect, the invention relates to a saccharide produced by expressing an wzz family protein (preferably fepE) in a gram-negative bacterium to produce a high molecular weight saccharide containing a medium-length O-antigen chain or a long O-antigen chain, said saccharide having an increase of at least 1, 2, 3, 4 or 5 repeating units compared to the corresponding wild-type O-polysaccharide. In one aspect, the invention relates to a saccharide produced by a cultured gram-negative bacterium that expresses (not necessarily overexpresses) an wzz family protein (e.g., wzzB) from a gram-negative bacterium to produce a high molecular weight saccharide containing a medium-length O-antigen chain or a long O-antigen chain, the saccharide having an increase of at least 1, 2, 3, 4, or 5 repeat units as compared to the corresponding wild-type O-antigen. For additional exemplary saccharides with an increased number of repeat units compared to the corresponding wild-type saccharide, see the description of O-polysaccharides and O-antigens below. The desired chain length is the chain length that results in improved or maximal immunogenicity in a given vaccine construct context.
In another embodiment, the saccharide comprises any one of the formulae selected from table 1, wherein the number n of repeat units in the saccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 66, 64, 66, 64, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 repeat units compared to the corresponding wild-type O-polysaccharide. See, e.g., table 24. Methods for determining saccharide length are known in the art. These methods include nuclear magnetic resonance, mass spectrometry and size exclusion chromatography, as described in example 13.
In a preferred embodiment, the invention relates to sugars produced in recombinant E.coli host cells, wherein the gene of an endogenous wzz O-antigen length modulator (e.g., wzzB) is deleted and replaced with a (second) wzz gene from a gram-negative bacterium heterologous to the recombinant E.coli host cell (e.g., Salmonella fepE) to produce high molecular weight sugars, such as lipopolysaccharides, containing a medium-length O-antigen chain or a long O-antigen chain. In some embodiments, the recombinant E.coli host cell comprises the wzz gene from Salmonella, preferably Salmonella enterica.
In one embodiment, the host cell comprises the heterologous gene for the wzz family protein in a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene comprising a wzz family protein in the form of an integrated gene in the chromosomal DNA of the host cell. Methods for stably expressing plasmid vectors in E.coli host cells and methods for integrating heterologous genes into the chromosome of E.coli host cells are known in the art. In one embodiment, the host cell comprises the heterologous gene for the O-antigen in the form of a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for the O-antigen in the form of an integrated gene in the chromosomal DNA of the host cell. Methods for stably expressing plasmid vectors in E.coli host cells and Salmonella host cells are known in the art. Methods for integrating heterologous genes into the chromosomes of E.coli host cells and Salmonella host cells are known in the art.
In one aspect, the recombinant host cell is cultured in a medium comprising a carbon source. Carbon sources for culturing E.coli are known in the art. Exemplary carbon sources include sugar alcohols, polyols, aldohexoses, or ketoses, including but not limited to arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinic acid, and methylamine. In a preferred embodiment, the culture medium comprises glucose. In some embodiments, the culture medium comprises a polyol or an aldol sugar, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose as a carbon source. All carbon sources may be added to the medium before the start of the culture, or may be added stepwise or continuously during the culture.
Exemplary media for recombinant host cells include those selected from KH2PO4,K2HPO4,(NH4)2SO4Sodium citrate, Na2SO4Aspartic acid, glucose, MgSO4,FeSO4-7H2O,Na2MoO4-2H2O,H3BO3,CoCl2-6H2O,CuCl2-2H2O,MnCl2-4H2O,ZnCl2And CaCl2-2H2O, or a combination thereof. Preferably, the medium comprises KH2PO4,K2HPO4,(NH4)2SO4Sodium citrate, Na2SO4Aspartic acid, glucose, MgSO4,FeSO4-7H2O,Na2MoO4-2H2O,H3BO3,CoCl2-6H2O,CuCl2-2H2O,MnCl2-4H2O,ZnCl2And CaCl2-2H2O。
The medium used herein may be solid or liquid, synthetic (i.e., artificial) or natural, and may contain sufficient nutrients for culturing the recombinant host cells. Preferably, the culture medium is a liquid culture medium.
In some embodiments, the culture medium may further comprise a suitable inorganic salt. In some embodiments, the culture medium may further comprise micronutrients. In some embodiments, the medium may further comprise a growth factor. In some embodiments, the medium may further comprise an additional carbon source. In some embodiments, the culture medium may further comprise suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources. Inorganic salts, micronutrients, growth factors and supplemental carbon sources suitable for use in culturing E.coli are known in the art.
In some embodiments, the medium may optionally comprise additional components such as peptones, N-Z amines, enzymatic soy hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins. In some embodiments, the culture medium does not comprise such additional components as peptones, N-Z amines, enzymatic soy hydrolysates, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins.
Illustrative examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates, and molasses; organic acids such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols such as glycerol, inositol, mannitol, and sorbitol.
In some embodiments, the medium further comprises a nitrogen source. Suitable nitrogen sources for the cultivation of E.coli are known in the art. Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts and other nitrogen containing materials including pure or crude amino acid preparations, meat extracts, peptones, fish meal, fish hydrolysates, corn steep liquor, casein hydrolysates, soy cake hydrolysates, yeast extracts, dry yeast, ethanol-yeast distillates, soy flour, cottonseed flour, and the like.
In some embodiments, the medium comprises an inorganic salt. Illustrative examples of suitable inorganic salts include, but are not limited to, potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten, and other trace elements and salts of phosphoric acid.
In some embodiments, the culture medium includes a suitable growth factor. Illustrative examples of suitable micronutrients, growth factors, and the like include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine hydrochloride, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6, 8-lipoic acid, folic acid, vitamin B, in the form of a pure or partially purified compound or in natural materials 12Other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p-or r-aminobenzoic acid, nicotinamide, nitriloacetate, and the like. The amount can be determined empirically by one skilled in the art based on methods and techniques known in the art
In another embodiment, the modified sugars described herein (as compared to the corresponding wild-type sugars) are produced synthetically, e.g., in vitro. Synthetic production or synthesis of sugars can help avoid cost and time intensive production processes. In one embodiment, the saccharide is synthetically synthesized from appropriately protected monosaccharide intermediates, e.g., by using a sequential glycosylation strategy or a combination of sequential glycosylation and [3+2] blocking synthetic strategies. For example, thioglycoside and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, the sugars synthesized synthetically in vitro have the same structure as those produced by recombinant means, such as by manipulation of the wzz family proteins described above.
The sugars produced (by recombinant or synthetic means) include structures derived from any e.coli serotype, including, for example, any of the following e.coli serotypes: o (e.g., O1 and O1), O (e.g., O: K and O: K), O (e.g., O5 and O5 (strain 180/C)), O (e.g., O: K; K and O: K), O (e.g., O18A, O18 and O18B), O (e.g., O23), O (e.g., O25 and O25), O (e.g., O and O45rel), O73)), O (e.g., O, 73, O, strain 73, O, C, O, C, O, C, O, C, O, 73, O, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, 0111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O156, O163, O175, O166, O175, O172, O185, O175, O166, O175, O172, O175, O168, O175, O166, O175, O185, O175, O166, O168, O175, O166, O172, O175, O168, O175, O166, O175, O164, O168, O175, O185, O166, O185, O168, O164, O168, O166, O175, O185, O175, O164, O166, O175, O168, O164, O168, O166, O175, O168, O164, O166, O168, O166, O168, O166, O168, O175, O168, O166, O168, O164, O168, O166, O168, O166, O168, O164, O168, O164, O168, O175, O168, O175, O168, O175, O168, O166, O168, O175, O168, O166, O168, O175, O168, O166, O168, O175, O168, O164.
The individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugate) by methods known in the art, such as dialysis, concentration procedures, diafiltration procedures, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultrafiltration, depth filtration and/or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE and hydrophobic interaction chromatography). Preferably, the polysaccharide is purified by a process comprising tangential flow filtration.
As further described herein, the purified polysaccharides can be activated (e.g., chemically activated) to enable them to react (e.g., directly with a carrier protein or through a linker such as an eTEC spacer), and then incorporated into glycoconjugates of the invention, as further described herein.
In a preferred embodiment, the saccharide of the invention is derived from an e.coli serotype, wherein the serotype is O25 a. In another preferred embodiment, the serotype is O25 b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.
As used herein, any serotype listed above is meant to encompass the repeating unit structures (O-units, described below) known in the art and which are unique to the corresponding serotype. For example, the term "O25 a" serotype (also referred to in the art as serotype "O25") is intended to encompass the serotype of formula O25 shown in table 1. As another example, the term "O25 b" serotype is intended to encompass the serotype of formula O25b shown in table 1.
As used herein, unless otherwise indicated, serotypes are herein generically referred to, such that, for example, the term "O18" is generically referred to encompass O18A, formula O18ac, formula 18a1, formula O18B, and formula O18B 1.
As used herein, the term "O1" refers broadly to a class that encompasses the formula including the generic term "O1" in the name of the formula according to table 1, such as any of formula O1A, formula O1a1, formula O1B, and formula O1C, each of which is shown in table 1. Thus, "O1 serotype" broadly refers to a serotype encompassing any one of formula O1A, formula O1a1, formula O1B, and formula O1C.
As used herein, the term "O6" generally refers to the class of formulae that includes the generic term "O6" in the names of formulae according to table 1, such as any of formulae O6: K2, K13, K15, and O6: K54, each of which is shown in table 1. Thus, "O6 serotype" broadly refers to a serotype encompassing any of O6: K2, K13, K15, and O6: K54.
Other examples of terms that generally refer to the class that includes the generic formula in the formula name according to table 1 include: "O4", "O5", "O18" and "O45".
As used herein, the term "O2" refers to the formula O2 shown in table 1. The term "O2O-antigen" is meant to encompass the saccharides of formula O2 shown in table 1.
As used herein, O-antigen from the serotypes listed above refers to a saccharide encompassing the formula labeled with the name of the corresponding serotype. For example, the term "O25B O-antigen" is meant to encompass the saccharides of formula O25B shown in table 1.
As another example, the term "O1O-antigen" generally refers to saccharides of the formula (such as formula O1A, formula O1a1, formula O1B, and formula O1C, each of which is shown in table 1) that are encompassed by the term "O1".
As another example, the term "O6O-antigen" refers broadly to saccharides that encompass the formula (such as the formula O6: K2; the formula O6: K13; the formula O6: K15 and the formula O6: K54), each of which is shown in Table 1, inclusive of the term "O6".
B.O polysaccharide
As used herein, the term "O-polysaccharide" refers to any structure that includes an O-antigen, provided that the structure does not include intact cells or lipid a. For example, in one embodiment, the O-polysaccharide comprises a lipopolysaccharide, wherein lipid a is not bound. The step of removing lipid a is known in the art, e.g. comprises heat treatment with addition of acid. An exemplary process includes treatment with 1% acetic acid at 100 ℃ for 90 minutes. This process is combined with a process of separating the removed lipid a. An exemplary method of isolating lipid a includes ultracentrifugation.
In one embodiment, an O-polysaccharide refers to a structure consisting of an O-antigen, in which case the O-polysaccharide is synonymous with the term O-antigen. In a preferred embodiment, O-polysaccharide refers to a structure comprising repeating units of an O-antigen, without a core sugar. Thus, in one embodiment, the O-polysaccharide does not include the E.coli R1 core moiety. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R2. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R3. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R4. In another embodiment, the O-polysaccharide does not include the E.coli K12 core moiety. In another preferred embodiment, O-polysaccharide refers to a structure comprising an O-antigen and a core sugar. In another embodiment, an O-polysaccharide refers to a structure that includes an O-antigen, a core sugar, and a KDO moiety.
Methods for purifying O-polysaccharides, including core oligosaccharides, from LPS are known in the art. For example, after purification of LPS, the purified LPS may be hydrolyzed by heating in 1% (v/v) acetic acid for 90 minutes at 100 degrees celsius, followed by ultracentrifugation at 142,000x g for 5 hours at 4 degrees celsius. The supernatant containing the O-polysaccharide was freeze dried and stored at 4 ℃. In certain embodiments, it is described that deletion of the capsule synthesis gene allows for simple purification of the O-polysaccharide.
The O-polysaccharide may be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid a from LPS. Other embodiments may include the use of hydrazine as an agent for the preparation of the O-polysaccharide. The preparation of LPS may be achieved by methods known in the art.
In certain embodiments, O-polysaccharides purified from wild-type, modified or attenuated gram-negative bacterial strains that express (do not necessarily overexpress) Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In a preferred embodiment, the O-polysaccharide chains are purified from gram-negative bacterial strains that express (and do not necessarily over-express) wzz protein for use as vaccine antigens (as a conjugate or composite vaccine).
In one embodiment, the molecular weight of the O-polysaccharide is increased by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 71-fold, 72-fold, 73 times, 74 times, 75 times, 76 times, 77 times, 78 times, 79 times, 80 times, 81 times, 82 times, 83 times, 84 times, 85 times, 86 times, 87 times, 88 times, 89 times, 90 times, 91 times, 92 times, 93 times, 94 times, 95 times, 96 times, 97 times, 98 times, 99 times, 100 times or more. In a preferred embodiment, the molecular weight of the O-polysaccharide is increased at least 1-fold and at most 5-fold compared to the corresponding wild-type O-polysaccharide. In another embodiment, the molecular weight of the O-polysaccharide is increased at least 2-fold and at most 4-fold compared to the corresponding wild-type O-polysaccharide. The increase in the molecular weight of the O-polysaccharide compared to the corresponding wild-type O-polysaccharide is preferably associated with an increase in the number of O-antigen repeating units. In one embodiment, the increase in molecular weight of the O-polysaccharide is due to the wzz family protein.
In one embodiment, the O-polysaccharide has an increased molecular weight compared to a corresponding wild-type O-polypeptide of about 1kDa,2kDa,3kDa,4kDa,5kDa,6kDa,7kDa,8kDa,9kDa,10kDa,11kDa,12kDa,13kDa,14kDa,15kDa,16kDa,17kDa,18kDa,19kDa,20kDa,21kDa,22kDa,23kDa,24kDa,25kDa,26kDa,27kDa,28kDa,29kDa,30kDa,31kDa,32kDa,33kDa,34kDa,35kDa,36kDa,37kDa,38kDa,39kDa,40kDa,41kDa,42kDa,43kDa,44kDa,45kDa,46kDa,47kDa,48kDa,49kDa,50, 51kDa,52kDa,53kDa,54kDa,55kDa,56kDa,57kDa,58kDa,59kDa,60kDa,61kDa,62kDa,63kDa,66 kDa,65kDa,66kDa,69 kDa,71kDa,73 kDa,74kDa,75kDa,76kDa,77kDa,78kDa,79kDa,80kDa,81kDa,82kDa,83kDa,84kDa,85kDa,86kDa,87kDa,88kDa,89kDa,90kDa,91kDa,92kDa,93kDa,94kDa,95kDa,96kDa,97kDa,98kDa,99kDa,100kDa or more. In one embodiment, the molecular weight of the O-polysaccharide of the invention is increased by at least 1kDa and at most 200kDa compared to the corresponding wild-type O-polysaccharide. In one embodiment, the molecular weight is increased by at least 5kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 15kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 30kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 90 kDa. In one embodiment, the molecular weight is increased by at least 12kDa and at most 85 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 70 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 60 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 50 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 49 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 48 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 47 kDa. In one embodiment, the molecular weight is increased by at least 10kDa and at most 46 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 45 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 44 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 43 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 42 kDa. In one embodiment, the molecular weight is increased by at least 20kDa and at most 41 kDa. This increase in the molecular weight of the O-polysaccharide compared to the corresponding wild-type O-polysaccharide is preferably associated with an increase in the number of O-antigen repeating units. In one embodiment, the increase in molecular weight of the O-polysaccharide is due to the wzz family protein. See, for example, table 21.
In another embodiment, the O-polysaccharide comprises any one of the formulae selected from table 1, wherein the number of repeat units n in the O-polysaccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 repeat units compared to the corresponding wild-type O-polysaccharide. See, for example, table 21.
C.O-antigen
The O-antigen is part of the Lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria. The O-antigen is located on the cell surface and is a variable cellular component. The variability of the O-antigen provides the basis for serotyping of gram-negative bacteria. Current E.coli serotyping protocols include O-polysaccharide 1 to 181.
O-antigens comprise oligosaccharide repeat units (O-units), the wild-type structure of which usually contains 2-8 residues from a variety of sugars. The O-units of exemplary E.coli O-antigens are shown in Table 1, see also FIG. 9A-FIG. 9C and FIG. 10A-FIG. 10B.
In one embodiment, the saccharide of the invention may be an oligosaccharide unit. In one embodiment, the saccharide of the invention is a repeating oligosaccharide unit of the relevant serotype. In such embodiments, the sugar may comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.
In one embodiment, the saccharide of the invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically obtained by synthesis or hydrolysis of polysaccharides. In such embodiments, the sugar may comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.
Preferably, all of the saccharides in the immunogenic compositions of the invention and the invention are polysaccharides. Because of the presence of epitopes on the surface of antigens, high molecular weight polysaccharides can induce certain antibody immune responses. Isolation and purification of high molecular weight polysaccharides is preferred for use in the conjugates, compositions and methods of the invention.
In some embodiments, the number of repeating O units in each individual O-antigen polymer (and thus the length and molecular weight of the polymer chains) is dependent on the wzz chain regulator (an inner membrane protein). Different wzz proteins were assigned different ranges of modal lengths (4 to >100 repeat units). The term "modal length" refers to the number of repeating O-units. Gram-negative bacteria typically have two different Wzz proteins that confer two different OAg modal chain lengths, one longer and one shorter. Expression (not necessarily over-expression) of wzz family proteins (e.g., wzzB) in gram-negative bacteria may allow manipulation of the length of the O-antigen to alter or bias bacterial production of O-antigens over certain length ranges and enhance production of high yields of high molecular weight lipopolysaccharides. In one embodiment, "short" modal length as used herein refers to a low number of repeating O-units, e.g., 1 to 20. In one embodiment, "long" modal length, as used herein, refers to a number of repeating O-units that exceeds 20 and is at most 40. In one embodiment, "very long" modal length as used herein refers to more than 40 repeating O-units.
In one embodiment, the produced saccharide is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 repeat units compared to the corresponding wild-type O-polysaccharide.
In another embodiment, the saccharide of the invention is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 67, 69, 72, 71, 73, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 repeat units compared to the corresponding wild type O-polysaccharide. See, for example, table 21. Methods for determining the length of a saccharide are known in the art. These methods include nuclear magnetic resonance, mass spectrometry and size exclusion chromatography, as described in example 13.
Methods for determining the number of repeat units in a saccharide are also known in the art. For example, the number of repeating units (or "n" in a formula) can be calculated by dividing the molecular weight of the polysaccharide (excluding the molecular weight of the core sugar or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure (e.g., shown in table 1) in the corresponding formula, which can be theoretically calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in the formula is known in the art. For example, the molecular weight of the repeat unit of formula O25b is about 862 Da. For example, the molecular weight of the repeat unit of formula O1a is about 845 Da. For example, the molecular weight of the repeat unit of formula O2 is about 829 Da. For example, the molecular weight of the repeat unit of formula O6 is about 893 Da. The molecular weight of the carrier protein and the ratio of protein to polysaccharide are considerations for the calculation when determining the number of repeat units in the conjugate. As defined herein, "n" refers to the number of repeating units in the polysaccharide molecule (shown in parentheses in table 1). As is known in the art, in biological macromolecules, repeating structures may be interspersed with regions of imperfect repetition, such as missing branches. In addition, it is known in the art that polysaccharides isolated and purified from natural sources such as bacteria may be heterogeneous in size and branching. In this case, n may represent the mean or median of n for the molecules in the population.
In one embodiment, the O-polysaccharide has at least one O-antigen repeating unit increased as compared to the corresponding wild-type O-polysaccharide. The repeating units of the O-antigen are shown in Table 1. In one embodiment, the O-polysaccharide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 76, 77, 78, 79, 77, 79, 73, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more total repeating units. Preferably, the saccharide has a total of at least 3 and at most 80 repeat units. In another embodiment, the O-polysaccharide is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 67, 69, 72, 71, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units.
In one embodiment, the saccharide comprises an O-antigen, wherein n in any of the O-antigen formulae (e.g., the formulae shown in table 1 (see also fig. 9A-9C and 10A-10B)) is an integer of at least 1,2,3,4,5,10,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40 and at most 200,100,99,98,97,96,95,94,93,92,91,90,89,88,87,86,81,80,79,78,77,76,75,74,73,72,71,70,69,68,67,66,65,60,59,58,57,56,55,54,53,52,51, or 50. Any minimum value and any maximum value may be combined to define a range. Exemplary ranges include, for example, at least 1 and up to 1000; at least 10 to at most 500; and at least 20 up to 80, preferably up to 90. In a preferred embodiment, n is at least 31 and at most 90. In a preferred embodiment, n is from 40 to 90, more preferably from 60 to 85.
In one embodiment, the saccharide comprises an O-antigen, wherein n in any one of the O-antigen formulae is at least 1 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 5 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 10 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 25 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 50 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 75 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 100 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 125 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 150 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 175 and at most 200. In one embodiment, n in any of the O-antigen formulae is at least 1 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 5 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 10 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 25 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 50 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 75 and at most 100. In one embodiment, n in any of the O-antigen formulae is at least 1 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 5 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 10 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 20 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 25 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 30 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 40 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 50 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 30 and at most 90. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 85. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 75. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 70. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 60. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 50. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 49. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 48. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 47. In one embodiment, n in any of the O-antigen formulae is at least 35 and at most 46. In one embodiment, n in any of the O-antigen formulae is at least 36 and at most 45. In one embodiment, n in any of the O-antigen formulae is at least 37 and at most 44. In one embodiment, n in any of the O-antigen formulae is at least 38 and at most 43. In one embodiment, n in any of the O-antigen formulae is at least 39 and at most 42. In one embodiment, n in any of the O-antigen formulae is at least 39 and at most 41.
For example, in one embodiment, n in the saccharide is 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89 or 90, most preferably 40. In another embodiment, n is at least 35 and at most 60. For example, in one embodiment, n is any one of 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59, and 60, preferably 50. In another preferred embodiment, n is at least 55 and at most 75. For example, in one embodiment, n is 55,56,57,58,59,60,61,62,63,64,65,66,67,68 or 69, most preferably 60.
Carbohydrate structures can be determined by methods and tools known in the art (e.g., NMR, including 1D, 1H, and/or 13C, 2D TOCSY, DQF-COSY, NOESY, and/or HMQC)
In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight between 5kDa and 400 kDa. In other such embodiments, the saccharide has a molecular weight between 10kDa and 400 kDa; between 5kDa and 400 kDa; between 5kDa and 300 kDa; between 5kDa and 200 kDa; between 5kDa and 150 kDa; between 10kDa and 100 kDa; between 10kDa and 75 kDa; between 10kDa and 60 kDa; between 10kDa and 40 kDa; between 10kDa and 100 kDa; between 10kDa and 200 kDa; between 15kDa and 150 kDa; between 12kDa and 120 kDa; between 12kDa and 75 kDa; between 12kDa and 50 kDa; between 12 and 60 kDa; between 35kDa and 75 kDa; between 40kDa and 60 kDa; between 35kDa and 60 kDa; between 20kDa and 60 kDa; between 12kDa and 20 kDa; or between 20kDa and 50 kDa. In yet another embodiment, the polysaccharide has a molecular weight between 7kDa and 15 kDa; between 8kDa and 16 kDa; between 9kDa and 25 kDa; between 10kDa and 100 kDa; between 10kDa and 60 kDa; between 10kDa and 70 kDa; between 10kDa and 160 kDa; between 15kDa and 600 kDa; between 20kDa and 1000 kDa; between 20kDa and 600 kDa; between 20kDa and 400 kDa; between 30kDa and 1,000 kDa; between 30kDa and 60 kDa; between 30kDa and 50kDa or between 5kDa and 60 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.
As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by Size Exclusion Chromatography (SEC) in combination with a multi-angle laser light scattering detector (MALLS).
During normal purification, the size of the polysaccharide is slightly reduced. In addition, as described herein, the polysaccharide may be subjected to sieving techniques prior to conjugation. Mechanical or chemical sieving may be employed. The chemical hydrolysis may be carried out using acetic acid. Mechanical sieving can be performed using high pressure homogenizing shear. The above molecular weight ranges refer to the polysaccharide purified prior to conjugation (e.g., prior to activation).
Table 1: escherichia coli serogroup/serotype and O-unit fraction
Figure BDA0003622971360000901
Figure BDA0003622971360000911
Figure BDA0003622971360000921
Figure BDA0003622971360000931
Figure BDA0003622971360000941
Figure BDA0003622971360000951
Figure BDA0003622971360000961
Figure BDA0003622971360000971
Figure BDA0003622971360000981
Figure BDA0003622971360000982
beta-D-6 dman Hep2Ac is 2-O-acetyl-6-deoxy- β -D-mannose-heptopyranosyl.
Figure BDA0003622971360000983
beta-D-Xulf is beta-D-threo-pentofuranosyl.
D. Core oligosaccharide
In wild-type E.coli LPS, the core oligosaccharide is located between lipid A and the outer region of the O-antigen. More specifically, the core oligosaccharide is the portion of the polysaccharide comprising the bond between the O-antigen and lipid A in wild-type E.coli. This linkage comprises a ketoglycoside linkage between the hemiketal functionality of the innermost 3-deoxy-d-manno-oct-2-ketonic acid (KDO)) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows a high degree of similarity between wild-type E.coli strains. Which usually contain a limited number of sugars. The core oligosaccharide comprises an internal core region and an external core region.
More specifically, the inner core is composed primarily of L-glycerol-D-manno-heptose (heptose) and KDO residues. The kernel is highly conserved. KDO residues include KDO of the formula:
Figure BDA0003622971360000991
the outer region of the core oligosaccharide shows more variation than the inner core region, and the difference in this region distinguishes five chemical types in e.coli: r1, R2, R3, R4 and K-12. See fig. 24, which illustrates the general structure of the carbohydrate backbone of five exo-oligosaccharides of known chemical type. HepI I is the last residue of the core oligosaccharide. Although all exo-oligosaccharides have a structural motif, have (hexoses)3A carbohydrate backbone and two side chain residues, but the order of the hexose in the backbone and the nature, position and attachment of the side chain residues may vary. The structures of R1 and R4 exo-oligosaccharides are highly similar, differing only by a single β -linked residue.
In the art, the core oligosaccharides of wild-type e.coli are divided into five different chemical classes based on the structure of the distal oligosaccharides: escherichia coli R1, Escherichia coli R2, Escherichia coli R3, Escherichia coli R4 and Escherichia coli K12.
In a preferred embodiment, the compositions described herein comprise a glycoconjugate in which the O-polysaccharide comprises a core oligosaccharide bound to an O-antigen. In one embodiment, the composition induces an immune response against at least any one of core e.coli chemotype e.coli R1, e.coli R2, e.coli R3, e.coli R4, and e.coli K12. In another embodiment, the composition induces an immune response against at least two core E.coli chemotypes. In another embodiment, the composition induces an immune response against at least three core E.coli chemotypes. In another embodiment, the composition induces an immune response against at least four core E.coli chemotypes. In another embodiment, the composition induces an immune response against all five core escherichia coli chemotypes.
In another preferred embodiment, the compositions described herein comprise glycoconjugates in which the O-polysaccharide does not comprise a core oligosaccharide bound to the O-antigen. In one embodiment, such a composition induces an immune response against at least any one of core e.coli chemotype e.coli R1, e.coli R2, e.coli R3, e.coli R4, and e.coli K12, although the glycoconjugate has an O-polysaccharide that does not include a core oligosaccharide.
The E.coli serotype can be characterized according to one of five chemical types. Table 2 lists typical serotypes characterized by chemical type. The serotypes in bold represent the serotypes most commonly associated with the core chemotypes shown. Thus, in a preferred embodiment, the composition induces an immune response against at least any one of the core escherichia coli chemotypes escherichia coli R1, escherichia coli R2, escherichia coli R3, escherichia coli R4, and escherichia coli K12, including an immune response against any one of the respective escherichia coli serotypes.
TABLE 2 core chemotypes and related E.coli serotypes
Figure BDA0003622971360001001
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the chemical form R1, e.g., selected from the group consisting of saccharides having the formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O8, formula O18, formula O9, formula O13, formula O20, formula O21, formula O91, and formula O163, wherein n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an e.coli R1 core moiety, e.g., as shown in figure 24.
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the chemical form R1, e.g., selected from the group consisting of saccharides having the formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O18, formula O13, formula O20, formula O21, formula O91, and formula O163, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an e.coli R1 core moiety in the saccharide.
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the chemical form R2, e.g., selected from the group consisting of saccharides having the formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, wherein n is from 1 to 100, preferably from 31 to 100, more preferably from 35 to 90, and most preferably from 35 to 65. In some embodiments, the saccharide in the composition further comprises an e.coli R2 core moiety, e.g., as shown in figure 24.
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the chemical form R3, e.g., selected from the group consisting of saccharides having the formula O25b, formula O15, formula O153, formula O21, formula O17, formula O11, formula O159, formula O22, formula O86, and formula O93, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an e.coli R3 core moiety, e.g., as shown in figure 24.
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the chemical form R4, e.g., selected from the group consisting of saccharides having the formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, wherein n is from 1 to 100, preferably from 31 to 100, more preferably from 35 to 90, and most preferably from 35 to 65. In some embodiments, the saccharide in the composition further comprises an e.coli R4 core moiety, e.g., as shown in figure 24.
In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having the K-12 chemistry (e.g., selected from the group consisting of a saccharide having the formula O25b and a saccharide having the formula O16), wherein n is 1 to 1000, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E.coli K-12 core moiety, e.g., as shown in FIG. 24.
In some embodiments, the sugar comprises a core sugar. Thus, in one embodiment, the O-polysaccharide further comprises a core portion of E.coli R1. In another embodiment, the O-polysaccharide further comprises an E.coli R2 core moiety. In another embodiment, the O-polysaccharide further comprises a core portion of E.coli R3. In another embodiment, the O-polysaccharide further comprises a core portion of E.coli R4. In another embodiment, the O-polysaccharide further comprises a core portion of E.coli K12.
In some embodiments, the saccharide does not include a core saccharide. Thus, in one embodiment, the O-polysaccharide does not include the core portion of E.coli R1. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R2. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R3. In another embodiment, the O-polysaccharide does not include the core portion of E.coli R4. In another embodiment, the O-polysaccharide does not include the E.coli K12 core moiety.
E. Conjugated O-antigens
Chemical linkage of the O-antigen or preferably the O-polysaccharide to the protein carrier can enhance the immunogenicity of the O-antigen or O-polysaccharide. However, variability in polymer size represents a practical challenge for production. In commercial applications, the size of the saccharide can affect compatibility with different conjugation synthesis strategies, homogeneity of the product, and immunogenicity of the conjugate. Controlling the expression of Wzz family protein chain length regulators by manipulating the O-antigen synthesis pathway allows the production of O-antigen chains of desired length in a variety of gram-negative bacterial strains, including E.coli.
In one embodiment, the purified saccharide is chemically activated to produce an activated saccharide capable of reacting with the carrier protein. Once activated, each saccharide is conjugated to a carrier protein to form a conjugate, i.e., a glycoconjugate. As used herein, the term "glycoconjugate" refers to a saccharide covalently linked to a carrier protein. In one embodiment, the saccharide is directly linked to the carrier protein. In another embodiment, the sugar is linked to the protein through a spacer/linker.
Conjugates can be prepared by a protocol in which the carrier is bound to the O-antigen at one site or along multiple sites of the O-antigen, or by a protocol in which at least one residue of the core oligosaccharide is activated.
In one embodiment, each saccharide is conjugated to the same carrier protein.
If the protein carrier of two or more saccharides in a composition is the same, the saccharides may be conjugated to the same carrier protein molecule (e.g., a carrier molecule having two or more different saccharides conjugated thereto).
In a preferred embodiment, the saccharides are each individually conjugated to different molecules of the protein carrier (each protein carrier molecule having only one type of saccharide bound to it). In such embodiments, the saccharide is considered to be conjugated to the carrier protein alone.
Chemical activation of the saccharide and subsequent conjugation to the carrier protein can be achieved by the activation and conjugation methods disclosed herein. After the polysaccharide has been bound to the carrier protein, the glycoconjugate is purified (enriched in terms of the amount of polysaccharide-protein conjugate) by a variety of techniques. These include concentration/diafiltration operations, precipitation/elution, column chromatography and depth filtration. After purification of the individual glycoconjugates, they are complexed to formulate the immunogenic compositions of the invention.
And (4) activating. The invention also relates to a composition as described hereinActivated polysaccharides produced in embodiments wherein the polysaccharide is activated with a chemical agent to produce reactive groups for conjugation to a linker or carrier protein. In some embodiments, the saccharide of the invention is activated and then conjugated to a carrier protein. In some embodiments, the degree of activation does not significantly reduce the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not cleave the polysaccharide backbone. In some embodiments, the degree of activation does not significantly affect the degree of conjugation, such as by a carrier protein (such as CRM)197) As measured by the number of modified lysine residues (as determined by amino acid analysis). For example, in some embodiments, the degree of activation does not significantly increase the number of modified lysine residues in the carrier protein (as determined by amino acid analysis) by a factor of 3 compared to the number of modified lysine residues in the carrier protein of a conjugate of the reference polysaccharide having the same degree of activation. In some embodiments, the degree of activation does not increase the level of unconjugated free saccharide. In some embodiments, the degree of activation does not decrease the optimal sugar/protein ratio.
In some embodiments, the activated saccharide has a percentage of activation, wherein the moles of thiol per saccharide repeat unit of the activated saccharide is between 1% and 100%, such as between 2% and 80%, between 2% and 50%, between 3% and 30%, and between 4% and 25%. The degree of activation is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, not less than 20%, not less than 30%, not less than 40%, not less than 50%, not less than 60%, not less than 70%, not less than 80%, not less than 90%, or not less than 100%. Preferably, the degree of activation is at most 50%, more preferably at most 25%. In one embodiment, the degree of activation is at most 20%. Any minimum value and any maximum value may be combined to define a range.
In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then conjugated to a carrier protein (preferably CRM) either directly or via a spacer (linker) group197Or tetanusToxin).
For example, the spacer can be cystamine or cysteamine to produce a thiolated polysaccharide that can be conjugated to a carrier via a thioether linkage that is activated at the carrier protein with maleimide (e.g., using N- [ Y-maleimide butyroyloxy acyloxy) ]Succinimidyl ester (GMBS)) or haloacetylated carrier proteins (e.g. using iodoacetamides, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), iodoacetic acid N-succinimidyl ester (SIA) or 3- [ bromoacetamido [)]Succinimidyl propionate (SBAP)) after reaction. In one embodiment, a cyanate ester (optionally manufactured by CDAP chemistry) is coupled with hexamethylene diamine or adipic Acid Dihydrazide (ADH), and the amino-derived saccharide is coupled with a carrier protein (e.g., CRM) via a carboxyl group on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry197) And (4) conjugation.
Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Conjugation may involve a carbonyl linker, which may be formed by reaction of the free hydroxyl group of the saccharide with CDI, followed by reaction with the protein to form a carbamate linkage. This may involve reducing the anomeric end to a primary hydroxyl group, optionally protecting/deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein (CDI chemistry).
And (3) molecular weight. In some embodiments, the glycoconjugate comprises a saccharide having a molecular weight between 10kDa and 2,000 kDa. In other embodiments, the saccharide has a molecular weight between 50kDa and 1,000 kDa. In other embodiments, the saccharide has a molecular weight between 70kDa and 900 kDa. In other embodiments, the saccharide has a molecular weight between 100kDa and 800 kDa. In other embodiments, the saccharide has a molecular weight between 200kDa and 600 kDa. In a further embodiment, the saccharide has a molecular weight of 100kDa to 1000 kDa; 100kDa to 900 kDa; 100kDa to 800 kDa; 100kDa to 700 kDa; 100kDa to 600 kDa; 100kDa to 500 kDa; 100kDa to 400 kDa; 100kDa to 300 kDa; 150kDa to 1,000 kDa; 150kDa to 900 kDa; 150kDa to 800 kDa; 150kDa to 700 kDa; 150kDa to 600 kDa; 150kDa to 500 kDa; 150kDa to 400 kDa; 150kDa to 300 kDa; 200kDa to 1,000 kDa; 200kDa to 900 kDa; 200kDa to 800 kDa; 200kDa to 700 kDa; 200kDa to 600 kDa; 200kDa to 500 kDa; 200kDa to 400 kDa; 200kDa to 300 kDa; 250kDa to 1,000 kDa; 250kDa to 900 kDa; 250kDa to 800 kDa; 250kDa to 700 kDa; 250kDa to 600 kDa; 250kDa to 500 kDa; 250kDa to 400 kDa; 250kDa to 350 kDa; 300kDa to 1,000 kDa; 300kDa to 900 kDa; 300kDa to 800 kDa; 300kDa to 700 kDa; 300kDa to 600 kDa; 300kDa to 500 kDa; 300kDa to 400 kDa; 400kDa to 1,000 kDa; 400kDa to 900 kDa; 400kDa to 800 kDa; 400kDa to 700 kDa; 400kDa to 600 kDa; 500kDa to 600 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by single-ended conjugation. In another embodiment, glycoconjugates having such molecular weights are produced by Reductive Amination Chemistry (RAC) prepared in aqueous buffer. Any integer within any of the above ranges is considered an embodiment of the present disclosure.
In some embodiments, the glycoconjugates of the invention have a molecular weight between 400kDa and 15,000 kDa; between 500kDa and 10,000 kDa; between 2,000kDa and 10,000 kDa; between 3,000kDa and 8,000 kDa; or between 3,000kDa and 5,000 kDa. In other embodiments, the glycoconjugate has a molecular weight between 500kDa and 10,000 kDa. In other embodiments, the glycoconjugate has a molecular weight between 1,000kDa and 8,000 kDa. In other embodiments, the glycoconjugate has a molecular weight between 2,000kDa and 8,000kDa or between 3,000kDa and 7,000 kDa. In yet another embodiment, the glycoconjugates of the invention have a molecular weight between 200kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200kDa and 10,000 kDa; between 200kDa and 7,500 kDa; between 200kDa and 5,000 kDa; between 200kDa and 3,000 kDa; between 200kDa and 1,000 kDa; between 500kDa and 20,000 kDa; between 500kDa and 15,000 kDa; between 500kDa and 12,500 kDa; between 500kDa and 10,000 kDa; between 500kDa and 7,500 kDa; between 500kDa and 6,000 kDa; between 500kDa and 5,000 kDa; between 500kDa and 4,000 kDa; between 500kDa and 3,000 kDa; between 500kDa and 2,000 kDa; between 500kDa and 1,500 kDa; between 500kDa and 1,000 kDa; between 750kDa and 20,000 kDa; between 750kDa and 15,000 kDa; between 750kDa and 12,500 kDa; between 750kDa and 10,000 kDa; between 750kDa and 7,500 kDa; between 750kDa and 6,000 kDa; between 750kDa and 5,000 kDa; between 750kDa and 4,000 kDa; between 750kDa and 3,000 kDa; between 750kDa and 2,000 kDa; between 750kDa and 1,500 kDa; between 1,000kDa and 15,000 kDa; between 1,000kDa and 12,500 kDa; between 1,000kDa and 10,000 kDa; between 1,000kDa and 7,500 kDa; between 1,000kDa and 6,000 kDa; between 1,000kDa and 5,000 kDa; between 1,000kDa and 4,000 kDa; between 1,000kDa and 2,500 kDa; between 2,000kDa and 15,000 kDa; between 2,000kDa and 12,500 kDa; between 2,000kDa and 10,000 kDa; between 2,000kDa and 7,500 kDa; between 2,000kDa and 6,000 kDa; between 2,000kDa and 5,000 kDa; between 2,000kDa and 4,000 kDa; or between 2,000kDa and 3,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein. In another embodiment, glycoconjugates having such molecular weights are produced by Reductive Amination Chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by Reductive Amination Chemistry (RAC) prepared in DMSO.
In yet another embodiment, the glycoconjugates of the invention have a molecular weight between 1,000kDa and 20,000 kDa; between 1,000kDa and 15,000 kDa; between 2,000kDa and 10,000 kDa; between 2000kDa and 7,500 kDa; between 2,000kDa and 5,000 kDa; between 3,000kDa and 20,000 kDa; between 3,000kDa and 15,000 kDa; between 3,000kDa and 12,500 kDa; between 4,000kDa and 10,000 kDa; between 4,000kDa and 7,500 kDa; between 4,000kDa and 6,000 kDa; or between 5,000kDa and 7,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by Reductive Amination Chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by Reductive Amination Chemistry (RAC) prepared in DMSO. In another embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein.
In yet another embodiment, the glycoconjugates of the invention have a molecular weight between 5,000kDa and 20,000 kDa; between 5,000kDa and 15,000 kDa; between 5,000kDa and 10,000 kDa; between 5,000kDa and 7,500 kDa; between 6,000kDa and 20,000 kDa; between 6,000kDa and 15,000 kDa; between 6,000kDa and 12,500 kDa; between 6,000kDa and 10,000kDa or between 6,000kDa and 7,500 kDa.
The molecular weight of the glycoconjugates can be measured by SEC-MALLS. Any integer within any of the above ranges is considered an embodiment of the present disclosure. The glycoconjugates of the invention can also be characterized by the ratio of saccharide to carrier protein (weight/weight). In some embodiments, the ratio (w/w) of polysaccharide to carrier protein in the glycoconjugate is between 0.5 and 3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the ratio of sugar to carrier protein (w/w) is between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and 1.0, between 1.0 and 1.5, or between 1.0 and 2.0. In yet another embodiment, the ratio of sugar to carrier protein (w/w) is between 0.8 and 1.2. In a preferred embodiment, the ratio of polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is CRM197。
Glycoconjugates can also be distributed by their molecular size (K)d) To characterize. Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size Exclusion Chromatography (SEC) was used in gravity-fed columns to delineate the molecular size distribution of the conjugates. The large molecules that are expelled from the pores of the medium elute more rapidly than the small molecules. Fraction collector was used to collect column eluate. The fractions were tested colorimetrically by carbohydrate determination. To determine Kd, the column was calibrated to determine the fraction of molecules completely excluded (V)0)，(Kd0), and a score (V) representing the maximum retentioni)，(Kd1). Score (V) to a specified sample attributee) According to the expression Kd＝(Ve-Vo)/(Vi-V0) And KdAnd (4) correlating.
Free sugar. The glycoconjugates and immunogenic compositions of the invention can comprise free saccharide that is not covalently conjugated to a carrier protein but is still present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (i.e. non-covalently bound to, adsorbed to or encapsulated in or by) the glycoconjugate. In preferred embodiments, the glycoconjugate comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises up to about 20% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises up to about 15% free polysaccharide compared to the total amount of polysaccharide. In another preferred embodiment, the glycoconjugate comprises at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 8% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises up to about 6% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises up to about 5% free polysaccharide compared to the total amount of polysaccharide. See, for example, table 19, table 20, table 21, table 22, table 23, table 24, and table 25.
Covalently linked. In other embodiments, for every 5 to 10 saccharide repeat units, every 2 to 7 saccharide repeat units, every 3 to 8 saccharide repeat units, every4 to 9 saccharide repeat units, 6 to 11 saccharide repeat units, 7 to 12 saccharide repeat units, 8 to 13 saccharide repeat units, 9 to 14 saccharide repeat units, 10 to 15 saccharide repeat units, 2 to 6 saccharide repeat units, 3 to 7 saccharide repeat units, 4 to 8 saccharide repeat units, 6 to 10 saccharide repeat units, 7 to 11 saccharide repeat units, 8 to 12 saccharide repeat units, 9 to 13 saccharide repeat units, 10 to 14 saccharide repeat units, 10 to 20 saccharide repeat units, 4 to 25 saccharide repeat units or 2 to 25 saccharide repeat units, the conjugate comprising at least one covalent linkage between the carrier protein and the saccharide. In a common embodiment, the carrier protein is CRM197. In another embodiment, there is at least one linkage between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeating units of polysaccharide. In one embodiment, the carrier protein is CRM 197. Any integer within any of the above ranges is considered an embodiment of the present disclosure.
A lysine residue. Another way to characterize glycoconjugates of the invention is by a carrier protein (e.g., CRM)197) The number of lysine residues conjugated to the saccharide, which can be characterized as a series of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent linkage to the polysaccharide can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to produce the conjugate material. In preferred embodiments, the glycoconjugates of the invention have a degree of conjugation between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and 12. In one embodiment The glycoconjugates of the present invention have a degree of conjugation of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15. In a preferred embodiment, the glycoconjugates of the invention have a degree of conjugation between 4 and 7. In some such embodiments, the carrier protein is CRM197。
The frequency of attachment of the sugar chain to the lysine on the carrier protein is another parameter characterizing the glycoconjugates of the invention. For example, in some embodiments, there is at least one covalent linkage between the carrier protein and the polysaccharide for every 4 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeat units of the polysaccharide. In yet another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeat units of the polysaccharide.
O-acetylation. In some embodiments, the saccharide of the invention is O-acetylated. In some embodiments, glycoconjugates comprise a saccharide having a degree of O-acetylation of between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 90% and 100%, between 50% and 90%, between 60% and 90%, between 70% and 90%, or between 80% and 90%. In other embodiments, the degree of O-acetylation is ≥ 10%, ≥ 20%, ≥ 30%, ≥ 40%, > 50%, > 60%, > 70%, > 80% or ≥ 90% or about 100%. The percentage of O-acetylation refers to the percentage of a given sugar with respect to 100% (where each repeating unit is fully acetylated with respect to its acetylated structure).
In some embodiments, the glycoconjugates are prepared by reductive amination. In some embodiments, the glycoconjugate is a single-ended conjugated saccharide, wherein the saccharide is covalently bound directly to the carrier protein. In some embodiments, the glycoconjugate is covalently bound to the carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer.
And (4) reductive amination. In one embodiment, the saccharide is conjugated to the carrier protein by reductive amination (such as described in U.S. patent application publication nos. 2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO 2008/079653 and WO 2008/143709).
Reductive amination involves (1) oxidation of the saccharide, (2) reduction of the activated saccharide with a carrier protein to form a conjugate. The sugar is optionally hydrolyzed prior to oxidation. Mechanical or chemical hydrolysis may be employed. The chemical hydrolysis may be carried out using acetic acid.
The oxidation step may involve reaction with periodate. As used herein, the term "periodate" refers to periodate and periodic acid. The term also includes metaperiodate (IO)4 -) And normal periodate (IO)6 5-) And various periodates (e.g., sodium periodate and potassium periodate). In one embodiment, the polysaccharide is in the presence of metaperiodate, preferably sodium periodate (NaIO) 4) In the presence of oxygen. In another embodiment, the polysaccharide is oxidized in the presence of periodic acid, preferably periodic acid.
In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxyl radical compound, such as a piperidine-N-oxyl or pyrrolidine-N-oxyl compound, that selectively oxidizes a primary hydroxyl group in the presence of the oxidizing agent. In the reaction, the actual oxidant is the N-oxoammonium salt in the catalytic cycle. In one aspect, the stable nitroxyl or nitroxyl radical compound is a piperidine-N-oxyl compound or a pyrrolidine-N-oxyl compound. In one aspect, the stable nitroxyl or nitroxyl radical compound has a TEMPO (2,2,6, 6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5, 5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, the oxidizing agent is a molecule bearing an N-halogen moiety. In one aspect, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3, 5-trichloro-1, 3, 5-triazinane-2, 4, 6-trione, dibromoisocyanuric acid, 1,3, 5-tribromo-1, 3, 5-triazinane-2, 4, 6-trione, diiodoisocyanuric acid, and 1,3, 5-triiodo-1, 3, 5-triazinane-2, 4, 6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.
After the sugar oxidation step, the sugar is considered to be activated, hereinafter referred to as "activated". The activated saccharide and carrier protein may be freeze-dried (lyophilized) separately (discrete freeze-drying) or together (co-freeze-drying). In one embodiment, the activated saccharide and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are separately lyophilized.
In one embodiment, the lyophilization is performed in the presence of a non-reducing sugar, possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit.
The next step in the conjugation process is the reduction of the activated saccharide and carrier protein using a reducing agent to form a conjugate (so-called reductive amination). Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium borohydride or zinc borohydride in the presence of a Bronsted or Lewis acid), amine boranes such as pyridine borane, 2-methyl pyridine borane, 2, 6-diborane-methanol, dimethylamine-borane, tert-butylborane-BH 3(t-BuMe' PrN-BH3), benzylamine-BH 3 or 5-ethyl-2-methyl pyridine borane (PEMB), borane-pyridine or borohydride exchange resins. In one embodiment, the reducing agent is sodium cyanoborohydride.
In one embodiment, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, N-Bis (2-hydroxyethyl) glycine (Bicine) or HEPB, pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5), in another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvents can be used to reconstitute the activated polysaccharide and carrier protein that have been lyophilized.
At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which may be blocked with a suitable blocking agent. In one embodiment, the capping agent is sodium borohydride (NaBH)4). After conjugation (reduction and optional capping), the glycoconjugate (enriched in terms of the amount of polysaccharide-protein conjugate) can be purified by various techniques known to the skilled person. These include dialysis, concentration/diafiltration operations, tangential flow filtration precipitation/elution, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration. The glycoconjugates may be purified by diafiltration and/or ion exchange chromatography and/or size exclusion chromatography. In one embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugate is sterile filtered.
In a preferred embodiment, the glycoconjugate derived from an e.coli serotype is selected from any one of O25B, O1, O2 and O6, prepared by reductive amination. In a preferred embodiment, glycoconjugates derived from e.coli serotypes O25B, O1, O2 and O6 are prepared by reductive amination.
In one aspect, the invention relates to a conjugate comprising a carrier protein linked to a saccharide of formula O25B, e.g., CRM197From
Figure BDA0003622971360001121
Where n is any integer greater than or equal to 1. In preferred embodiments, n is an integer of at least 31,32,33,34,35,36,37,38,39,40 and up to 200,100,99,98,97,96,95,94,93,92,91,90,89,88,87,86,81,80,79,78,77,76,75,74,73,72,71,70,69,68,67,66,65,60,59,58,57,56,55,54,53,52,51, or 50. Any minimum value and any maximum value may be combined to define a range. Exemplary ranges include, for example, at least 1 and up to 1000; at least 10 and at most 500; and at least 20 up to 80. In a preferred embodiment, n is at least 31 and at most 90, more preferably 40 to 90, most preferably 60 to 85.
In another aspect, the invention relates to conjugates comprising a carrier protein (e.g., CRM) linked to a saccharide having any one of the following structures shown in table 1 197) (see also FIGS. 9A-9C and 10A-10B) where n is an integer greater than or equal to 1.
Without being bound by theory or mechanism, in some embodiments, stable conjugates are believed to require a level of saccharide antigen modification that is balanced with maintaining the structural integrity of key immunogenic epitopes of the antigen.
Activation and formation of aldehydes. In some embodiments, the saccharide of the invention is activated and results in the formation of an aldehyde. In such embodiments where the saccharide is activated, the percentage (%) of activation (or Degree of Oxidation (DO)) (see, e.g., example 31) refers to the number of moles of saccharide repeat units per mole of aldehyde of activated polysaccharide. For example, in some embodiments, the saccharide is activated by periodate oxidation of vicinal diols on the polysaccharide repeat units, resulting in the formation of aldehydes. During oxidation, varying the molar equivalents of sodium periodate relative to the sugar repeat units (meq) and temperature results in a change in the Degree of Oxidation (DO).
The concentrations of sugars and aldehydes are typically determined by colorimetric assays. An alternative reagent is the TEMPO (2,2,6, 6-tetramethylpiperidine 1-oxyl radical) -N-chlorosuccinimide (NCS) combination, which results in the formation of aldehydes from primary alcohol groups.
In some embodiments, the activated saccharide has a degree of oxidation wherein the number of moles of saccharide repeat units per mole of aldehyde of the activated saccharide is between 1 and 100, such as between 2 and 80, between 2 and 50, between 3 and 30, and between 4 and 25. The activation degree is at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, more than or equal to 20, more than or equal to 30, more than or equal to 40, more than or equal to 50, more than or equal to 60, more than or equal to 70, more than or equal to 80, more than or equal to 90 or about 100. Preferably, the Degree of Oxidation (DO) is at least 5 and at most 50, more preferably at least 10 and at most 25. In one embodiment, the degree of activation is at least 10 and at most 25. Any minimum value and any maximum value may be combined to define a range. The oxidation degree value can be expressed as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to one activated saccharide repeat unit of a total of 10 saccharide repeat units in the activated saccharide, in which case a DO value of 10 can be expressed as 10% activation.
In some embodiments, the conjugate prepared by reductive amination chemistry comprises a carrier protein and a saccharide, wherein the saccharide comprises a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula I, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D1, formula O63, formula O64, formula O65 (for example formula O65 (73-1 strain)), formula O65, formula O124, formula O114, formula O65, formula O685102, formula O65, formula O123, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O685103, formula O685126, formula O65, formula O685126, formula O65, formula O685103, formula O685126, formula O685103, formula O685126, formula O65, formula O685126, formula O65, formula, Formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187. In some embodiments, the saccharide in the conjugate comprises a formula wherein n is an integer from 1 to 1000, 5 to 1000, preferably from 31 to 100, more preferably from 35 to 90, most preferably from 35 to 65.
A single-end linked conjugate. In some embodiments, the conjugate is a single-ended conjugated saccharide, wherein the saccharide is covalently bound to a carrier protein at one end of the saccharide. In some embodiments, the single-end linked conjugated polysaccharide has a terminal sugar. For example, if one terminus of the polysaccharide (the terminal sugar residue) is covalently bound to the carrier protein, the conjugate is single-ended. In some embodiments, the conjugate is single-ended if the terminal saccharide residue of the polysaccharide is covalently bound to the carrier protein through a linker. Such linkers may include, for example, cystamine linker (a1), 3 ' -dithiobis (malonic acid dihydrazide) linker (a4), and 2,2 ' -dithio-N, N ' -bis (ethane-2, 1-diyl) bis (2- (aminooxy) acetamide) linker (a 6).
In some embodiments, the saccharide is conjugated to the carrier protein through a 3-deoxy-d-mannose-octa-2-ketosugar acid (KDO) residue to form a single-end linked conjugate. See, for example, example 26, example 27, example 28 and figure 17.
In some embodiments, the conjugate is preferably not a bioconjugate. The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen (e.g., an O antigen prepared in the context of a host cell (e.g., O25B)), where the host cell mechanism links (e.g., N-links) the antigen to the protein. Glycoconjugates include bioconjugates, as well as glycoantigen (e.g., oligo-and polysaccharides) -protein conjugates prepared by a means that does not require the preparation of the conjugate in a host cell, e.g., by conjugation of a protein to a saccharide.
Thiol-activated saccharides. In some embodiments, the saccharide of the invention is thiol-activated. In embodiments where the saccharide is activated by a thiol, the percent (%) of activation refers to the number of moles of thiol in each saccharide repeat unit of the activated polysaccharide. The concentrations of sugars and thiols are typically determined by an Ellman assay for thiol quantification. For example, in some embodiments, the sugar comprises activation of 2-keto-3-deoxyoctanoic acid (KDO) using an amine disulfide linker. See, for example, example 10 and figure 31. In some embodiments, the saccharide is covalently bound to the carrier protein through a divalent heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the saccharide and the carrier protein, resulting in a glycoconjugate referred to herein as a "thioether glycoconjugate". In some embodiments, the linker also provides carbamate and amide linkages, e.g., (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC). See, for example, example 21.
In some embodiments, the single-ended linked conjugate comprises a carrier protein and a saccharide, wherein the saccharide comprises a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula I, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D1, formula O63, formula O64, formula O65 (for example formula O65 (73-1 strain)), formula O65, formula O685103, formula O114, formula O685106, formula O65, formula O685106, formula O65, formula O685120, formula O103, formula O65, formula O103, formula O685106, formula O65, formula O103, formula O65, formula O685106, formula O65, formula O65, formula O103, formula O65, formula O685125, formula O65, formula O65, formula O103, formula O65, formula O65, formula O103, formula O65, formula O103, formula O65, formula O685125, formula O65, formula O103, formula O103, formula O103, formula O65, formula O103, formula O65, formula O103, formula O103, formula O65, formula O104, formula O103, formula O formula, Formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187. In some embodiments, the saccharide in the conjugate comprises a formula wherein n is an integer from 1 to 1000, 5 to 1000, preferably from 31 to 100, more preferably from 35 to 90, most preferably from 35 to 65.
For example, in one embodiment, a single-ended conjugate comprises a carrier protein and a saccharide having a structure selected from the group consisting of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, wherein n is an integer from 1 to 10.
eTEC conjugates
In one aspect, the invention generally relates to glycoconjugates comprising a saccharide derived from escherichia coli as described above covalently conjugated to a carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer (as described, for example, in U.S. patent 9517274 and international patent application publication WO2014027302, which are incorporated herein by reference in their entirety), including immunogenic panels comprising such glycoconjugatesCompounds, and methods for making and using such glycoconjugates and immunogenic compositions. The glycoconjugates comprise a saccharide covalently conjugated to a carrier protein through one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacers through a carbamate linkage, and wherein the carrier protein is covalently conjugated to the eTEC spacers through an amide linkage. The eTEC spacer comprises seven linear atoms (i.e., -C (O) NH (CH)2)2SCH2C (o) and provides stable thioether and amide linkages between the saccharide and the carrier protein.
The eTEC-linked glycoconjugates of the invention can be represented by the general formula (I):
Figure BDA0003622971360001171
wherein the atoms that make up the eTEC spacer are contained in a central box.
In the glycoconjugates of the present invention, the saccharide may be a polysaccharide or an oligosaccharide.
The carrier protein incorporated into the glycoconjugates of the invention is selected from the group of carrier proteins generally suitable for this purpose, as further described herein or known to those skilled in the art. In a particular embodiment, the carrier protein is CRM197。
In another aspect, the invention provides a method of preparing a glycoconjugate comprising a saccharide described herein conjugated to a carrier protein via an eTEC spacer, the method comprising the steps of: a) reacting a sugar with a carbonic acid derivative in an organic solvent to produce an activated sugar; b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more alpha-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α -haloacetamido groups of the activated carrier protein and/or (ii) a second capping reagent capable of capping unconjugated free thiol residues of the activated thiolated saccharide; thereby producing an eTEC linked glycoconjugate.
In a common embodiment, the carbonic acid derivative is 1,1 '-carbonyl-bis- (1,2, 4-triazole) (CDT) or 1, 1' -Carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). In a preferred embodiment, the thiolated saccharide is produced by reaction of the activated saccharide with a bifunctional symmetric thioalkylamine reagent, cystamine, or a salt thereof. Alternatively, the thiolated saccharide may be formed by reaction of an activated saccharide with cysteamine or a salt thereof. The eTEC-linked glycoconjugates produced by the methods of the invention can be represented by general formula (I).
In a common embodiment, the first capping agent is N-acetyl-L-cysteine, which reacts with unconjugated α -haloacetamide groups on lysine residues of the carrier protein to form S-carboxymethylcysteine (CMC) residues covalently linked to activated lysine residues through thioether linkages.
In other embodiments, the second capping reagent is Iodoacetamide (IAA), which reacts with the unconjugated free thiol group of the activated thiolated saccharide to provide a capped thioacetamide. Typically, step e) comprises capping with both the first and second capping agents. In certain embodiments, step e) comprises capping with N-acetyl-L-cysteine as the first capping reagent and IAA as the second capping reagent.
In some embodiments, the blocking step e) further comprises a reaction with a reducing agent, such as DTT, TCEP or mercaptoethanol, after the reaction with the first and/or second capping reagent.
The eTEC-linked glycoconjugates and immunogenic compositions of the invention may comprise free sulfhydryl residues. In some cases, the activated thiolated saccharide formed by the methods provided herein will include a plurality of free sulfhydryl residues, some of which may not be covalently conjugated to a carrier protein during the conjugation step. Such residual free thiol residues are blocked by reaction with a thiol-reactive blocking agent, such as Iodoacetamide (IAA), to block potentially reactive functional groups. Other thiol-reactive capping reagents, such as maleimide-containing reagents and the like, are also contemplated.
In addition, the eTEC-linked glycoconjugates and immunogenic compositions of the invention can comprise residual unconjugated carrier protein, which can comprise an activated carrier protein that has undergone modification during the capping process step.
In some embodiments, step d) further comprises providing the activated carrier protein comprising one or more α -haloacetamide groups prior to reacting the activated thiolated saccharide with the activated carrier protein. In a common embodiment, the activated carrier protein comprises one or more α -bromoacetamide groups.
In another aspect, the invention provides an eTEC-linked glycoconjugate comprising a saccharide described herein conjugated to a carrier protein via an eTEC spacer produced according to any of the methods disclosed herein.
In some embodiments, the carrier protein is CRM197And CRM197Covalent linkage to the polysaccharide via the eTEC spacer occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide.
For each aspect of the invention, in particular embodiments of the methods and compositions described herein, the eTEC-linked glycoconjugate comprises a saccharide described herein, such as a saccharide derived from e.
In another aspect, the invention provides a method of preventing, treating or ameliorating a bacterial infection, disease or disorder in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention, wherein the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a saccharide as described herein. In some embodiments, the saccharide is derived from e.
In some embodiments, the eTEC-linked glycoconjugate comprises a carrier protein and a saccharide, wherein the saccharide comprises a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula I, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D1, formula O63, formula O64, formula O65 (for example formula O65 (73-1 strain)), formula O65, formula O124, formula O114, formula O65, formula O685102, formula O65, formula O123, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O65, formula O685126, formula O685103, formula O685126, formula O65, formula O685126, formula O65, formula O685103, formula O685126, formula O685103, formula O685126, formula O65, formula O685126, formula O65, formula, Formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187. In some embodiments, the saccharide in the conjugate comprises a formula wherein n is an integer from 1 to 1000, 5 to 1000, preferably from 31 to 100, more preferably from 35 to 90, most preferably from 35 to 65.
The number of saccharide-conjugated lysine residues in the carrier protein can be characterized as a series of conjugated lysines. For example, in some embodiments of the immunogenic composition, CRM197May comprise 4 to 16 lysine residues of the 39 lysine residues covalently linked to the saccharide. Another way to express this parameter is about 10% to about 41% CRM197Lysine is covalently linked to a sugar. In other embodiments, CRM197May comprise 2 to 20 lysine residues of the 39 lysine residues covalently linked to the saccharide. Another way to express this parameter is about 5% to about 50% CRM197Lysine is covalently linked to a sugar.
In a common embodiment, the carrier protein is CRM197And CRM197Covalent linkage to the polysaccharide via the eTEC spacer occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide.
In other embodiments, the conjugate comprises at least one covalent linkage between the carrier protein and the saccharide for every 5 to 10 saccharide repeat units, every 2 to 7 saccharide repeat units, every 3 to 8 saccharide repeat units, every 4 to 9 saccharide repeat units, every 6 to 11 saccharide repeat units, every 7 to 12 saccharide repeat units, every 8 to 13 saccharide repeat units, every 9 to 14 saccharide repeat units, every 10 to 15 saccharide repeat units, every 2 to 6 saccharide repeat units, every 3 to 7 saccharide repeat units, every 4 to 8 saccharide repeat units, every 6 to 10 saccharide repeat units, every 7 to 11 saccharide repeat units, every 8 to 12 saccharide repeat units, every 9 to 13 saccharide repeat units, every 10 to 14 saccharide repeat units, every 10 to 20 saccharide repeat units, or every 4 to 25 saccharide repeat units.
In another embodiment, there is at least one linkage between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 repeat units of polysaccharide.
G. Carrier proteins
One component of the glycoconjugates of the invention is the carrier protein to which the saccharide is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be compatible with standard conjugation procedures.
One component of the conjugate is a carrier protein conjugated to an O-polysaccharide. In one embodiment, the conjugate comprises a carrier protein conjugated to a core oligosaccharide of an O-polysaccharide (see figure 24). In one embodiment, the conjugate comprises a carrier protein conjugated to an O-antigen of an O-polysaccharide.
The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be compatible with standard conjugation procedures.
In a preferred embodiment, the carrier proteins of the conjugate are independently selected from TT, DT mutants (such as CRM) 197) Any of haemophilus influenzae protein D, PhtX, PhtD, PhtDE fusions (especially those described in WO 01/98334 and WO 03/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin a or B of clostridium difficile and PsaA. In an embodiment, the carrier protein of the conjugate of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the conjugate of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the conjugate of the invention is PD (haemophilus influenzae protein D-see, e.g., EP 0594610B). In some embodiments, the carrier protein comprises poly (L-lysine) (PLL).
In a preferred embodiment, the sugar is CRM197And (4) protein conjugation. CRM197The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM197Generated by corynebacterium diphtheriae (c. diphtheriae) infected with non-toxigenic bacteriophage β 197tox-, which was generated by nitrosoguanidine mutagenesis of corynebacterium toxigenic β. CRM197The protein has the same molecular weight as diphtheria toxin, but differs therefrom by a single base change in the structural gene (guanine to adenine) (iv) a pharmaceutically acceptable salt thereof). This single base change results in an amino acid substitution of glutamic acid to glycine in the mature protein and eliminates the toxic properties of diphtheria toxin. CRM197The protein is a safe and effective T cell-dependent sugar transporter.
Thus, in some embodiments, the conjugates of the invention comprise CRM197As a carrier protein, wherein the saccharide is complexed with CRM197And (3) covalent linkage.
In a preferred embodiment, the carrier protein of the glycoconjugate is selected from the group consisting of: DT (diphtheria toxin), TT (tetanus toxoid) or fragment C, CRM of TT197(non-toxic but antigenically identical variants of diphtheria toxin), other DT mutants (such as CRM176, CRM228, CRM45 (Uchida et al J.biol. chem.218; 9-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM 107; and other mutations described by Nichols and Youle in genetic Engineered Toxins, Ed: Frankel, Maxel Dekker Inc, 1992; Glu-148 deletion or mutation to Asp, gin or Ser and/or Ala 158 or mutation to Gly, and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more residues in Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5917017 or US 64673; or fragments disclosed in US 5843711), pneumolysin (Streptococcus pneumoniae et al (1995) and Inlmn 2706; including for example, PLdBS 8613, GmbH 13-04081515, in a certain detoxification manner such as PLdBS 13, WO 24, PCT/EP2005/010258) or dPLY-formaldehyde, PhtX, including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00/37105 or WO 00/39299) and fusions of Pht proteins, such as PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334, WO 03/54007, WO2009/000826), OMPC (meningococcal outer membrane proteins-usually extracted from Neisseria meningitidis (N.meningidis) serogroup B-EP0372501), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae) protein D-see, e.g., EP 9405610B) or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP 04347), heat shock proteins (WO 272, WO 94/03208), pertussis proteins (WO 98/58668), EP 0471177), EP 71177, EP 715, PhtD-cells, Lymphokines, growth factors, or hormones (WO 9) 1/01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as the N19 protein (Baraldoi et al (2004) infection lmmun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron-uptake protein (WO 01/72337), toxin A or B of Clostridium difficile (C.difficile) (WO 00/61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (in particular non-toxic mutants thereof, such as exotoxin A with a substitution at glutamate 553 (Uchida Cameron DM, RJ Collier.1987.J.Bacteriol.169: 4967-4971)). Other proteins, such as ovalbumin, Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or purified protein derivatives of tuberculin (PPD) may also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in international patent application No. WO 2004/083251), escherichia coli LT, escherichia coli ST, and exotoxin a from pseudomonas aeruginosa.
In some embodiments, the carrier protein is selected from, for example, any one of: CRM 197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid or exotoxin a from pseudomonas aeruginosa; detoxified exotoxin a (epa) of pseudomonas aeruginosa, Maltose Binding Protein (MBP), flagellin, detoxified hemolysin a of staphylococcus aureus (s. aureus), aggregation factor a, aggregation factor B, cholera toxin B subunit (CTB), streptococcus pneumoniae pneumolysin and detoxified variants thereof, campylobacter jejuni (c. jejuni) AcrA, and campylobacter jejuni native glycoproteins. In one embodiment, the carrier protein is detoxified pseudomonas Exotoxin (EPA). In another embodiment, the carrier protein is not detoxified pseudomonas Exotoxin (EPA). In one embodiment, the carrier protein is a flagellin. In another embodiment, the carrier protein is not a flagellin.
In a preferred embodiment, the carrier proteins of the glycoconjugates are independently selected from the group consisting ofThe group consisting of: TT, DT mutants (such as CRM)197) Haemophilus influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO 01/98334 and WO 03/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin a or B of clostridium difficile and PsaA. In one embodiment, the carrier protein of the glycoconjugate of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the glycoconjugate of the invention is PD (haemophilus influenzae protein D-see, e.g., EP 0594610B).
In a preferred embodiment, the capsular saccharides of the invention are conjugated to CRM197And (4) protein conjugation. CRM197The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM197Generated by infection with Corynebacterium diphtheriae infected with the non-toxigenic bacteriophage β 197tox-, generated by nitrosoguanidine mutagenesis of toxigenic coryneform bacteriophage β (Uchida, T. et al 1971, Nature New Biology 233: 8-11). CRM197The protein has the same molecular weight as diphtheria toxin, but differs therefrom by a single base change in the structural gene (guanine to adenine). This single base change results in an amino acid substitution of glutamic acid to glycine in the mature protein and eliminates the toxic properties of diphtheria toxin. CRM197The protein is a safe and effective T cell-dependent sugar transporter. With respect to CRM197More details of and the production thereof can be found, for example, in US 5,614,382.
Thus, in a common embodiment, the glycoconjugates of the invention comprise CRM197As a carrier protein, wherein the capsular polysaccharide is CRM197And (3) covalent linkage.
H. Dosage of the composition
The dosage regimen may be adjusted to provide the best desired response. For example, a single dose of an e.coli-derived polypeptide or fragment thereof may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the situation. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is also to be understood that for any particular subject, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Determining the appropriate dosage and regimen for therapeutic protein administration is well known in the relevant art and, once the teachings disclosed herein are provided, will be understood to those skilled in the art to be included therein.
In some embodiments, the amount of the polypeptide derived from E.coli, or fragment thereof, in the composition can range from about 10 μ g to about 300 μ g per protein antigen. In some embodiments, the amount of polypeptides derived from E.coli, or fragments thereof, in the composition can range from about 20 μ g to about 200 μ g per protein antigen.
The amount of the one or more glycoconjugates in each dose is selected to induce an immune protective response in a typical vaccine without significant adverse side effects. Such amounts will vary depending on the particular immunogen used and the manner of presentation thereof.
The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on the total polysaccharide (conjugated and unconjugated) of the conjugate. For example, a glycoconjugate with 20% free polysaccharide will have about 80g of conjugated polysaccharide and about 20g of unconjugated polysaccharide in a 100g polysaccharide dose. The amount of glycoconjugate may vary depending on the e.coli serotype. The sugar concentration can be determined by uronic acid assay.
The "immunogenic amount" of the different polysaccharide components in the immunogenic composition can vary, and each can comprise about 1.0g, about 2.0g, about 3.0g, about 4.0g, about 5.0g, about 6.0g, about 7.0g, about 8.0g, about 9.0g, about 10.0g, about 15.0g, about 20.0g, about 30.0g, about 40.0pg, about 50.0pg, about 60.0pg, about 70.0pg, about 80.0pg, about 90.0pg, or about 100.0g of any particular polysaccharide antigen. Typically, for a given serotype, each dose will contain from 0.1g to 100g of polysaccharide, particularly from 0.5g to 20g, more particularly from 1g to 10g, even more particularly from 2g to 5 g. Any integer within any of the above ranges is considered an embodiment of the present invention. In one embodiment, each dose will contain 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 15g or 20g of polysaccharide for a given serotype.
Amount of carrier protein. Typically, each dose will comprise from 5g to 150g of carrier protein, particularly from 10g to 100g of carrier protein, more particularly from 15g to 100g of carrier protein, more particularly from 25g to 75g of carrier protein, more particularly from 30g to 70g of carrier protein, more particularly from 30g to 60g of carrier protein, more particularly from 30g to 50g of carrier protein, even more particularly from 40g to 60g of carrier protein. In one embodiment, the carrier protein is CRM197. In one embodiment, each dose will comprise about 25g, about 26g, about 27g, about 28g, about 29g, about 30g, about 31g, about 32g, about 33g, about 34g, about 35g, about 36g, about 37g, about 38g, about 39g, about 40g, about 41g, about 42g, about 43g, about 44g, about 45g, about 46g, about 47g, about 48g, about 49g, about 50g, about 51g, about 52g, about 53g, about 54g, about 55g, about 56g, about 57g, about 58g, about 59g, about 60g, about 61g, about 62g, about 63g, about 64g, about 65g, about 66g, about 67g,68g, about 69g, about 70g, about 71g, about 72g, about 73g, about 74g, or about 75g of the carrier protein. In one embodiment, the carrier protein is CRM197。
I. Adjuvant
In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two, or three adjuvants. The term "adjuvant" refers to a compound or mixture that enhances an immune response to an antigen. The antigen may be primarily as a delivery system, primarily as an immunomodulator, or have strong characteristics of both. Suitable adjuvants include those suitable for use in mammals including humans.
Examples of known suitable delivery system type adjuvants that may be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80(Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide and poly (D, L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.
In embodiments, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In preferred embodiments, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In embodiments, the immunogenic compositions disclosed herein comprise 0.1mg/mL to 1mg/mL or 0.2mg/mL to 0.3mg/mL of elemental aluminum in the form of aluminum phosphate. In embodiments, the immunogenic compositions disclosed herein comprise elemental aluminum in the form of about 0.25mg/mL aluminum phosphate. Examples of known suitable immunomodulatory adjuvants that can be used in humans include, but are not limited to, saponin extracts from the bark of the tree ajillate (Aquilla tree) (QS21, Quil a), TLR4 agonists such AS MPL (monophosphoryl lipid a), 3DMPL (3-O-deacylated MPL) or GLA-AQ, LT/CT mutants, cytokines such AS various interleukins (e.g. AS IL-2, IL-12) or GM-CSF, AS01, and the like.
Examples of known suitable immunomodulatory adjuvants with delivery and immunomodulatory properties that can be used in humans include, but are not limited to, ISCOMS (see, e.g.,
Figure BDA0003622971360001261
et al (1998) J.Leukocyte biol.64: 713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EM (which is a TLR4 agonist in combination with an oil-in-water emulsion).
For veterinary applications, including but not limited to animal experiments, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, abbreviated to nor-MDP), N-acetyl muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, abbreviated to MTP-PE) and RIBI, the RIBI contains three components extracted from bacteria: monophosphoryl lipid a, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in 2% squalene Tween 80 emulsion.
Other exemplary adjuvants that enhance the efficacy of the immunogenic compositions disclosed herein include, but are not limited to, (1) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components), for example (a) SAF, containing 10% squalane, 0.4% Tween 80, 5% pluronic blocking polymer L121, and thr-MDP, which are microfluidized into submicron emulsions or vortexed to produce emulsions of greater particle size, and (b) RIBI bi TMAdjuvant System (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components such as monophosphoryl lipid a (MPL), Trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS), preferably MPL + CWS (DETOX)TM) (ii) a (2) Saponin adjuvants, such as QS21, STIMULONTM(Cambridge Bioscience,Worcester,Mass.),
Figure BDA0003622971360001271
(Isconova, Sweden) or
Figure BDA0003622971360001272
(Commonwelth Serum Laboratories, Australia) may be used, or particles produced therefrom, such as ISCOMs (immune stimulating complexes) which may be free of additional detergents (e.g., WO 00/07621); (3) complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99/44636)), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF), and the like; (5) monophosphoryl lipid a (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB2220211, EP0689454) (see, e.g., WO 00/56358); (6)3dMPL in combination with, for example, QS21 and/or an oil-in-water emulsion (see, for example, EP0835318, EP0735898, EP 0761231); (7) polyoxyethylene ethers or esters (see, for example, WO 99/52549); (8) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (e.g. WO 01/21207), or polyoxyethylene in combination with at least one additional non-ionic surfactant such as octoxynol Alkyl ether or ester surfactants (e.g., WO 01/21152); (9) saponins and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO 00/62800); (10) immunostimulants and metal salt particles (see, e.g., WO 00/23105); (11) saponins and oil-in-water emulsions (e.g. WO 99/11241); (12) saponins (e.g. QS21) +3dMPL + IM2 (optionally + sterols) (e.g. WO 98/57659); (13) other substances used as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine-PE), and the like.
In an embodiment of the invention, the immunogenic compositions disclosed herein comprise CpG oligonucleotides as adjuvants. As used herein, a CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and thus the terms are used interchangeably unless otherwise indicated. The immunostimulatory CpG oligodeoxynucleotides comprise one or more immunostimulatory CpG motifs, which motifs are unmethylated cytosine-guanine dinucleotides, optionally within certain preferred base ranges. The methylation state of CpG immunostimulatory motifs generally refers to cytosine residues in dinucleotides. An immunostimulatory oligonucleotide comprising at least one unmethylated CpG dinucleotide is an oligonucleotide comprising a 5 'unmethylated cytosine linked to a 3' guanine by a phosphate bond and activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides that will activate the immune system through TLR9, but not as strongly as when one or more CpG motifs are unmethylated. The CpG immunostimulatory oligonucleotides may comprise one or more palindromic sequences, which in turn may comprise CpG dinucleotides. CpG oligonucleotides have been described in a number of issued patents, published patent applications, and other publications, including U.S. Pat. nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068.
In an embodiment of the invention, the immunogenic composition disclosed herein comprises any CpG oligonucleotide described on page 3 line 22 to page 12 line 36 of WO 2010/125480.
Different classes of CpG immunostimulatory oligonucleotides have been identified. These oligonucleotides are referred to as class A, class B, class C and class P and are described in more detail on page 3, line 22 to page 12, line 36 of WO 2010/125480. The methods of the invention encompass the use of these different classes of CpG immunostimulatory oligonucleotides.
VI I. nanoparticles
In another aspect, disclosed herein is an immunogenic complex comprising 1) a nanostructure; and 2) at least one pilus polypeptide antigen or fragment thereof. Preferably, the pilus polypeptide or fragment thereof is derived from E.coli pilus H (fimH). In a preferred embodiment, the pilus polypeptide is selected from any of the pilus polypeptides described above. For example, the pilus polypeptide may comprise any one of the amino acid sequences selected from SEQ ID NOs:1-10,18,20,21,23,24 and 26-29.
In some embodiments, the antigen is fused or conjugated to the exterior of the nanostructure to stimulate the onset of an adaptive immune response against the displayed epitope. In some embodiments, the immunogenic complex further comprises an adjuvant or other immunomodulatory compound attached to the exterior and/or encapsulated inside the cage to help tailor the type of immune response generated against each pathogen.
In some embodiments, the nanostructure comprises a single assembly comprising a plurality of identical first nanostructure-associated polypeptides.
In an alternative embodiment, the nanostructure comprises a plurality of modules (which comprise a plurality of identical first nanostructure-associated polypeptides) and a plurality of second modules, each second module comprising a plurality of identical second nanostructure-associated polypeptides.
Various nanostructure platforms can be used to produce the immunogenic compositions described herein. In some embodiments, the nanostructures employed are formed from multiple copies of a single subunit. In some embodiments, the nanostructures employed are formed from multiple copies of multiple different subunits.
The nanostructures are generally spherical and/or have rotational symmetry (e.g., having a 3-and 5-heavy axis), e.g., an icosahedral structure as exemplified herein.
In some embodiments, the antigen is presented on a self-assembling nanoparticle such as self-assembling nanostructures derived from Ferritin (FR), E2p, Q β and I3-01. E2p is a redesigned variant of the dihydroacyl acyltransferase from Bacillus stearothermophilus. I3-01 is an engineered protein that can self-assemble into ultrastable nanoparticles. The sequences of these protein subunits are known in the art. In a first aspect, disclosed herein are nanostructure-associated polypeptides comprising an amino acid sequence that is at least 75% identical in length to the amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOS:59-92, and is identical at least one identified interface position. Nanostructure-related polypeptides can be used, for example, to make nanostructures. Nanostructure-related polypeptides are designed based on their ability to self-assemble in pairs to form nanostructures, such as icosahedral nanostructures.
In some embodiments, the nanostructure comprises (a) a plurality of first modules, each first module comprising a plurality of identical first nanostructure-associated polypeptides, wherein the first nanostructure-associated polypeptides comprise the amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOS: 59-92; and (b) a plurality of second components, each second component comprising a plurality of identical second nanostructure-associated polypeptides, wherein the second nanostructure-associated polypeptides comprise an amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOS 59-92, and wherein the second nanostructure-associated polypeptide is different from the first nanostructure-associated polypeptide; wherein the plurality of first components non-covalently interact with the plurality of second components to form a nanostructure;
the nanostructures include a symmetrically repeating, non-natural, non-covalent polypeptide-polypeptide interface that orients the first and second components into a nanostructure, such as a nanostructure having icosahedral symmetry.
SEQ ID NOS 59-92 provide the amino acid sequences of exemplary nanostructure-associated polypeptides. Exemplary nanostructure-related polypeptides of SEQ ID NOs 59-92 have interfacial residue numbers ranging from 4-13 residues. In various embodiments, the nanostructure-associated polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in length to the amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOS:59-92, and is identical at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the identified interface positions (depending on the number of interface residues for a given nanostructure-associated polypeptide). In other embodiments, the nanostructure-associated polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in length to the amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOS:59-92, and is at least 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% identical at the identified interface positions. In yet another embodiment, the nanostructure-associated polypeptide comprises a nanostructure-associated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS 59-98.
In one non-limiting embodiment, the nanostructure-associated polypeptide can be modified to facilitate covalent linkage to a "cargo" of interest. In one non-limiting example, the nanostructure-associated polypeptides can be modified, such as by introducing various cysteine residues at defined positions (to facilitate linkage to one or more antigens of interest), such that the nanostructure of the nanostructure-associated polypeptides will provide a scaffold to provide a large number of antigens for vaccine delivery, resulting in an improved immune response.
In some embodiments, some or all of the native cysteine residues present in the nanostructure-associated polypeptide, but not intended for conjugation, may be mutated to other amino acids to facilitate conjugation at a defined position. In another non-limiting embodiment, the nanostructure-associated polypeptide can be modified by linkage (covalent or non-covalent) to a moiety to help facilitate "endosomal escape". For applications involving delivery of a molecule of interest to a target cell, such as targeted delivery, a critical step may be the escape from the endosome (a membrane-bound organelle that is the entry point for delivery of the vehicle into the cell). Endosomes mature into lysosomes, which degrade their contents. Thus, if the delivery vehicle does not somehow "escape" from the endosome before it becomes lysosomal, it will be degraded and unable to perform its function. There are a variety of lipids or organic polymers that disrupt endosomes and escape into the cytosol. Thus, in this embodiment, the nanostructure-associated polypeptide may be modified, for example by the introduction of cysteine residues, which would allow chemical conjugation of such lipids or organic polymers to the monomers or resulting assembly surfaces. In another non-limiting example, the nanostructure-associated polypeptide can be modified, for example, by the introduction of cysteine residues, which would allow chemical conjugation of fluorophores or other imaging agents, thereby allowing the nanostructure to be visualized in vitro or in vivo.
Surface amino acid residues on the nanostructure-associated polypeptide can be mutated to improve the stability or solubility of the protein subunit or assembled nanostructure. As known to those skilled in the art, if a nanostructure-associated polypeptide has significant sequence homology to an existing protein family, multiple sequence alignments of other proteins from that family can be used to guide selection of amino acid mutations at non-conserved positions that can increase protein stability and/or solubility, a process known as consensus protein design (9).
Surface amino acid residues on nanostructure-associated polypeptides can be mutated to positively charged (Arg, Lys) or negatively charged (Asp, Glu) amino acids to impart an overall positive or negative charge to the protein surface. In one non-limiting embodiment, surface amino acid residues on the nanostructure-associated polypeptide can be mutated to impart a high net charge to the inner surface of the self-assembled nanostructure. Such nanostructures can then be used to package or encapsulate cargo molecules having opposite net charges due to electrostatic interactions between the inner surface of the nanostructure and the cargo molecules. In one non-limiting embodiment, the surface amino acid residues on the nanostructure-associated polypeptide can be mutated primarily to arginine or lysine residues to impart a net positive charge to the inner surface of the self-assembled nanostructure. The solution comprising the nanostructure-associated polypeptide can then be mixed in the presence of a nucleic acid cargo molecule such as dsDNA, ssDNA, dsRNA, ssRNA, cDNA, miRNA, siRNA, shRNA, piRNA, or other nucleic acid, in order to encapsulate the nucleic acid inside the self-assembled nanostructure. Such nanostructures can be used, for example, to protect, deliver, or concentrate nucleic acids.
In one embodiment, the nanostructure has icosahedral symmetry. In this embodiment, the nanostructure can comprise 60 copies of the first nanostructure-associated polypeptide and 60 copies of the second nanostructure-associated polypeptide. In one such embodiment, the number of identical first nanostructure-associated polypeptides in each first component is different from the number of identical second nanostructure-associated polypeptides in each second component. For example, in one embodiment, the nanostructure comprises twelve first modules and twenty second modules; in this embodiment, each first component may, for example, comprise five copies of the same first nanostructure-associated polypeptide, while each second component may, for example, comprise three copies of the same second nanostructure-associated polypeptide. In another embodiment, the nanostructure comprises twelve first modules and thirty second modules; in this embodiment, each first component may, for example, comprise five copies of the same first nanostructure-associated polypeptide, and each second component may, for example, comprise two copies of the same second nanostructure-associated polypeptide. In yet another embodiment, the nanostructure comprises twenty first modules and thirty second modules; in this embodiment, each first component may, for example, comprise three copies of the same first nanostructure-associated polypeptide, and each second component may, for example, comprise two copies of the same second nanostructure-associated polypeptide. All of these embodiments are capable of forming synthetic nanomaterials with regular icosahedral symmetry.
Examples
In order that the invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. The following examples illustrate some embodiments of the invention.
Example 1: overview of the constructs
TABLE 3
Figure BDA0003622971360001331
Figure BDA0003622971360001341
Figure BDA0003622971360001351
Figure BDA0003622971360001361
Figure BDA0003622971360001371
All FimH constructs studied were monomeric proteins with the expected molecular weight.
TABLE 4
Figure BDA0003622971360001372
Figure BDA0003622971360001381
The expected molecular weight of the FimC-FimH complex is 53.1 kDa;
FimC has an expected molecular weight of 24 kDa.
Example 2: mammalian expression of the FimH lectin binding domain
The non-limiting example of the invention relates to the production of polypeptides derived from E.coli or fragments thereof in a HEK cell line. The yield is relatively high compared to expressing the polypeptide derived from E.coli or a fragment thereof in an E.coli host cell.
To achieve production of FimH variants from mammalian cells, the different heterologous signal sequences secreted by proteins and fragments were analyzed using the SignalP prediction algorithm. The wild-type FimH leader sequence was also analyzed. Prediction suggests that the wild-type FimH leader sequence may have an effect on secreted FimH variants in mammalian cells, however, it is predicted that secreted variants are cleaved at residue W20 of full-length wild-type FimH (see SEQ ID NO:1) rather than at residue F22 of full-length wild-type FimH (see SEQ ID NO: 1). The hemagglutinin signal sequence was predicted to be non-functional. The murine IgK signal sequence is predicted to yield the F22N terminus of SEQ ID NO 1, or F1 residue of the mature protein.
Based on these analyses, DNA was synthesized and constructs were generated recombinantly to express FimH lectin binding domains with wild-type FimH leader sequences. Constructs expressing FimH lectin binding domains with mIgK signal sequences were also prepared. Affinity purification tags, such as His tags, are introduced to the C-terminus of E.coli-derived polypeptides or fragments thereof to facilitate purification.
The expression plasmid was transfected into HEK host cells, EXPI293 mammalian cells.
Successfully expressed was a polypeptide derived from E.coli or a fragment thereof. For example, the preferred N-terminal processing of the pSB01892 FimHdscG construct using the mIgK signal sequence fused to the maturation origin of FimH at F22 was confirmed by MS. For the lectin domain construct pSB01878, the processing was considered correct and mass spectral data supported this.
For the native FimH leader, NO preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) has been shown.
The pSB01877 and pSB01878 constructs were in pcDNA3.1(+) mammalian expression vectors. Cells were diluted and subsequently used for 20ml transfection. 1ug/ml DNA was used for each construct and cells in 125ml flasks were transfected using the Expifactamine protocol. After 72 hours, cell viability was still good, thus allowing expression to continue for 96 hours. Samples were taken at 72 hours and 10. mu.l each was run on SDS PAGE gels to check for expression.
After 96 hours, the conditioned medium was harvested and 0.25ml of Nickel Excel resin was added and the O/N (batch binding O/N) was bound in portions at 4 ℃ under rotation. Eluted in TrisCl pH8.0, NaCl, imidazole. See fig. 4.
The expected mass of pSB01878 is consistent with N-terminal F22. Glycosylation is present at 1 or 2 sites (per deamidation of N-D +1 mass).
And constructing glycosylation mutants. See, e.g., pSB02081, pSB02082, pSB02083, pSB02088, and pSB 02089. The glycosylation mutants express the target polypeptide. The results are shown in FIG. 5.
FimH lectin domain lock mutants were also constructed. See, e.g., pSB 02158. The expression result of the pSB02158 construct is shown in FIG. 6B.
Fluorescence polarization assay using 0.5 picomolar fluorescein conjugated aminophenyl-mannopyranoside (APMP). The measurement was performed at 300RPM for 64 hours at room temperature. The results are shown in FIG. 6C.
Example 3: mammalian expression of the FimH/C complex, pSB01879 and pSB01880
To generate the FimH/C complex, a dual expression construct of FimC under the EF1 α promoter and FimH with a wild-type or mIgK signal peptide was prepared. These were cloned into the pBudCE4.1 mammalian expression vector (ThermoFisher) and the C-terminal His tag was added to the FimC. FimC variants were designed for secretion using the mIgK signal peptide, as this led to a positive prediction based on the SignalP assay that produced G37 FimC as the first residue of the mature protein.
More specifically, these constructs were designed to have the FimC fragment under the EF1 promoter in the vector pbudce4.1 and to insert the FimH fragment under the CMV promoter in the same vector. The vector pBudCE4.1 is an expression vector from Thermo Fisher with 2 promoters for expression in mammalian cells. The FimC fragment insert (pSB01881 insert) was subcloned by digestion with NotI and XhoI and subcloned into the pbudce4.1 vector at the same site. These were plated on 2XYT zeocin 50ug/ml plates. Colonies were inoculated into 2XYT containing zeocin at 50ug/ml, grown overnight at 37 ℃ and plasmids prepared. These plasmids were digested with NotI and XhoI to examine the inserts, and the insert size of all colonies was approximately 722 bp.
pSB01881 was digested with HindIII and BamHI and the pSB01879 insert and pSB01880 insert DNA were digested with HindIII and BamHI. These fragments were gel separated, subcloned into pSB01881 vector, and plated on 2XYTzeo50 ug/ml plates. Colonies from each were inoculated into 2xYT zeo50ug/ml, grown overnight at 37 ℃, plasmids were prepared, digested with NotI and XhoI to test FimC inserts, and with HindIII and BamHI to test FimH inserts. All clones had inserts of the expected size at both cloning sites. The pSB01879-1 and pSB01880-1 clones were subsequently used for expression.
The FimH/FimC complex has also been demonstrated to be expressed in EXPI293 cells. Expression can be optimized by switching promoters such as EF1 α, CAG, Ub, Tub or others.
For the native FimH leader, NO preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) has been shown.
Exemplary results for SignalP 4.1(DTU Bioinformatics) used for signal peptide prediction are shown below. It is expected that the additional signal peptide will produce a preferred Phe N-terminus at position 1 of the mature FimH polypeptide or fragment thereof. The following is only a representative sample set of 4 common signal sequences.
The following signal peptide sequence is predicted to produce a preferred Phe N-terminus at position 1 of the mature FimH polypeptide or fragment thereof:
TABLE 5
Figure BDA0003622971360001411
The following signal peptide sequence was not predicted to produce the preferred Phe N-terminus at position 1 of the mature FimH polypeptide or fragment thereof:
TABLE 6
Figure BDA0003622971360001412
TABLE 7 SignalP 4.1 for prediction
Figure BDA0003622971360001413
Figure BDA0003622971360001421
Figure BDA0003622971360001431
Figure BDA0003622971360001441
Example 4: mammalian expression of complementary fusions of FimH and donor strands of FimG peptides
Several splice lengths were tested. Recombinant expression was made using these linkers to fuse FimH to the N-terminal FimG peptide in wild-type FimH and the mIgK signal peptide fused to F22 of FimH.
FimH donor strand complementary FimG constructs have also been shown to have robust expression in EXPI293 cells.
For the native FimH leader, NO preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) was shown.
For the donor strand complementary constructs, the oligonucleotides were designed to produce basic constructs containing various linkers and FimG peptides in pcdna3.1 (+). A unique BstEII site was incorporated at residue G294V 295T 296, according to the numbering of FimH SEQ ID NO 1. The same BstEII site was incorporated into the linker to generate the basic construct.
The basic structure of pSB01882-01895 was constructed. The primers were used to PCR amplify pcDNA3.1(+) with ACCUPRIME PFX DNA polymerase (Thermo Fisher), the PCR product was digested with NdeI (in the CMV promoter) and BamHI and cloned into NdeI and BamHI digested pcDNA3.1(+) and gel separated to remove fragments.
Another transient transfection was performed with pSB01877,01878,01879,01880,01885 and 01892 and EXPI293 cells as controls.
Constructs pSB01882 to pSB01895 were used for transient transfection expression testing of EXPI293 cells from Thermo Fisher, according to the manufacturer's protocol. See FIG. 3, which shows the results after loading 10ul conditioned medium for 72 hours of expression in 20mL EXPI293 cells; high levels of expression were observed; the FimH/FimC complex exhibits the following expression from pSB01879 and pSB01880 constructs; a20 ml batch of conditioned medium was combined with Nickel Excel, washed 40CV and eluted in imidazole.
Additional FimH-donor strand complementary constructs were prepared. See, e.g., pSB02198, pSB02199, pSB02200, pSB02304, pSB02305, pSB02306, pSB02307, pSB02308 constructs. Expression of the pSB2198 FimH dscG episome construct is shown in figure 7. The pSB2198 FimH dscG latanomutant produced 12mg/L from transient expression.
According to Vi-CELL XR 2.04(Beckman Coulter, Inc.), the following is observed (the actual CELL type used for expression is HEK CELLs):
TABLE 8
Figure BDA0003622971360001451
Figure BDA0003622971360001461
Example 5: molecular weight fragments and treated signal peptides
TABLE 9
Figure BDA0003622971360001462
Figure BDA0003622971360001471
Figure BDA0003622971360001481
pSB02083
Analysis of Fragment 21-188 Intact proteins
Length of 168aa 188aa
Molecular weight 18063.42 20290.02m.w.
1 microgram ═ 55.361 picomole 49.285 picomole
Extinction coefficient of molecule 24420 35800
1A (280) accurate to: 0.74mg/ml 0.57mg/ml
1mg/ml of A [280 ]] 1.35AU 1.76AU
Isoelectric point 6.81 6.29
Charge at pH 7 -0.48 -2.47
pSB02198
Figure BDA0003622971360001491
pSB02307
Figure BDA0003622971360001492
Example 6: the N-terminal alpha-amino group of Phe1 (numbering according to SEQ ID NO: 2) in the mature protein of FimH provides a key polar recognition for D-mannose
Without being bound by theory or mechanism, it has been suggested that correct signal peptide cleavage just prior to Phe1 (numbering according to SEQ ID NO: 2) of the FimH mature protein is important for expression of functional FimH proteins. Changes at the N-terminal alpha-amino group (such as by adding an amino acid at the N-terminus before Phe1 of FimH protein) can eliminate hydrogen bonding interactions with the O2-, O5-, and O6-atoms of D-mannose and introduce steric repulsion with D-mannose, thereby blocking mannose binding. Our experimental observations confirmed that the addition of an additional Gly residue before Phe1 of SEQ ID NO 2 resulted in NO mannose binding detected.
After analyzing the crystal structure of FimH bound to D-mannose, the following results were observed: the N-terminal alpha-amino group of Phe1 of FimH according to the numbering of SEQ ID NO. 2 as well as the side chain of Asp54 and Gln133 of FimH according to the numbering of SEQ ID NO. 2 provide a key polar recognition motif for D-mannose, these mutations and alterations of polar interaction leading to NO mannose binding.
Example 7: the side chain of Phe1 in FimH did not interact directly with D-mannose, but was buried inside FimH, indicating that Phe1 could be substituted by other residues such as aliphatic hydrophobic residues (Ile, Leu or Val)
Analysis of the crystal structure of FimH complexed with D-mannose and its analogues (e.g. PDB ID: 1QUN) showed that the side chain of Phe1 (numbering according to SEQ ID NO: 2) did not interact directly with D-mannose, but stabilized the binding pocket by stacking its aromatic rings with the side chains of Val56, Tyr95, Gln133 and Phe144 (numbering according to SEQ ID NO: 2).
Replacement of the N-terminal residue in place of Phe can stabilize the FimH protein, accommodate mannose binding, and allow for proper signal peptide cleavage. Such residues may be identified by suitable methods known in the art, such as by visual inspection of the crystal structure of FimH, or more quantitative selection using computational protein design software, such as BioLuminate TM[BioLuminate,Schrodinger LLC,New York,2017],Discovery StudioTM[Discovery Studio Modeling Environment,Dassault Systèmes,San Diego,2017],MOETM[Molecular Operating Environment,Chemical Computing Group Inc.,Montreal,2017]And RosettaTM[Rosetta,University of Washington,Seattle,2017]. Illustrative examples are shown in fig. 9A-9C. The replacement amino acid may be an aliphatic hydrophobic amino acid (e.g., Ile, Leu, and Val). FIG. 11 depicts a computational mutagenesis scan of Phe1 with other amino acids with aliphatic hydrophobic side chains (e.g., Ile, Leu, and Val) that can stabilize the FimH protein and accommodate mannose binding.
Example 8: mutation of Asn7 numbered according to SEQ ID NO 2 in the FimH protein can remove the putative N-glycosylation site and prevent deamidation without affecting the binding of mannose, mAb21 or mAb 475.
Overexpression of secreted E.coli FimH by mammalian cell lines can result in N-linked glycosylation at residue Asn7 (numbering according to SEQ ID NO: 2). In addition, the residue Asn7 is exposed to solvent, followed by a Gly residue, making it very susceptible to deamidation.
Analysis of the crystal structure of FimH complexed with D-mannose and its analogues (e.g., PDB ID:1QUN) indicates that Asn7 is more than one point from the mannose binding site
Figure BDA0003622971360001512
And mutations at this site do not affect mannose binding. Thus, mutation of Asn7 to other amino acids (e.g., Ser, Asp, and gin) can effectively remove the putative N-glycosylation site and prevent deamidation.
Example 9: escherichia coli and salmonella enterica (s. enterica) strains
Clinical strains and derivatives are listed in table 10. Other reference strains include: O25K5H1, a clinical O25a serotype strain; and salmonella Typhimurium (s. enterica serovar Typhimurium) strain LT 2.
Gene knockouts were constructed in e.coli strains, removing the targeted open reading frame, but leaving short scar sequences.
For simplicity, the hydrolyzed O-antigen chains and core sugars are subsequently denoted as O-polysaccharides (OPS).
Figure BDA0003622971360001511
Figure BDA0003622971360001521
Example 10: oligonucleotide primers for cloning wzzB, fepE and O-antigen gene clusters
TABLE 11 oligonucleotide primers
Figure BDA0003622971360001522
TABLE 11 oligonucleotide primers
Figure BDA0003622971360001531
Example 11: plasmids
Plasmid vectors and subclones are listed in table 12. PCR fragments containing various E.coli and Salmonella wzzZB and fepE genes were amplified from purified genomic DNA and subcloned into Invitrogen
Figure BDA0003622971360001532
Among the high copy number plasmids provided in the Blunt cloning kit, fig. 12A-fig. 12B. The plasmid is based on a pUC replicon. Primers P3 and P4 were used to amplify the E.coli wzzB gene with its native promoter and were designed to bind to regions in the proximal and distal genes (annotated in Genbank MG1655 NC-000913.3) encoding UDP-glucose-6-dehydrogenase and phosphoribosyladenine nucleotidohydrolase, respectively. PCR fragments containing the Salmonella fepE gene and promoter were amplified using the aforementioned primers. Similar E.coli fepE primers were designed based on the available Genbank genomic sequence or the internally generated complete genomic data (in the case of GAR2401 and O25K5H 1). The low copy number plasmid pBAD33 was used to express the O-antigen biosynthesis genes under the control of the arabinose promoter. Plasmids (Table 12) were first engineered to facilitate cloning (by the Gibson method) of long PCR fragments amplified using universal primers homologous to the 5 'promoter and 3' 6-phosphogluconate dehydrogenase (gnd) gene. The pBAD33 subclone containing the O25B biosynthetic operon was shown in FIGS. 12A-12B.
TABLE 12 plasmids
Figure BDA0003622971360001541
Example 12: o-antigen purification
Treating the fermentation broth with acetic acid to a final concentration of 1-2% (final pH)Is 4.1). Extraction and defatting of OAg was achieved by heating the acid treated broth to 100 ℃ for 2 hours. At the end of the acid hydrolysis, the batch was cooled to ambient temperature and 14% NH was added4OH to a final pH of 6.1. The neutralized broth was centrifuged and the concentrate was collected. Adding CaCl in sodium phosphate to the concentrate2The resulting slurry was incubated at room temperature for 30 minutes. The solids were removed by centrifugation and the concentrate was concentrated 12-fold using a 10kDa membrane and then diafiltered twice against water. The retentate containing OAg is then purified using a carbon filter. The carbon filtrate was diluted 1:1(v/v) with 4.0M ammonium sulfate. The final concentration of ammonium sulfate was 2M. The ammonium sulfate treated carbon filtrate was further purified with a membrane using 2M ammonium sulfate as the running buffer. OAg was collected in the flow-through. For long OAg, the HIC filtrate was concentrated and the buffer was exchanged with water (20 permeate volumes) using a 5kDa membrane. For short (native) OAg polysaccharides, MWCO is further reduced to improve yield.
Example 13: o25b Long O-antigen with CRM197Conjugation of (2)
Oxidation with periodate to generate a first set of long chain O25b polysaccharide-CRM 197Conjugate, followed by conjugation using Reductive Amination Chemistry (RAC) (table 14). Conjugate variants with three activation levels (low, medium and high) by varying the oxidation level. Lyophilized activated polysaccharide reconstituted in DMSO medium with lyophilized CRM using sodium cyanoborohydride as reducing agent197Reacting to produce the conjugate. The conjugation reaction was carried out at 23 ℃ for 24 hours, then blocked with sodium borohydride for 3 hours. After the conjugation quenching step, the conjugate was purified by ultrafiltration/diafiltration using 100K MWCO regenerated cellulose membrane using 5mM succinate/0.9% NaCl, pH 6.0. The conjugate was subjected to final filtration using a 0.22 μm membrane.
Unless specifically stated otherwise, the conjugates disclosed throughout the following examples include a core sugar moiety.
1.1. Long O-antigen expression conferred by heterologous polymerase chain length modulators
The initial E.coli strain construction was focused on the O25 serotype. The goal was to overexpress heterologous wzzB or fepE genes to see if they confer longer chain length in O25 wzzB knockout strains. First, blood isolates were screened by PCR to identify strains of subtypes O25a and O25 b. Next, the strains were screened for sensitivity to ampicillin. A single ampicillin sensitive O25b isolate GAR2401 was identified into which a wzzB deletion was introduced. Similarly, a deletion of wzzB was made in O25a strain O25K5H 1. For genetic complementation of these mutations, the wzzB genes from GAR2401 and O25K5H1 were subcloned into a high copy PCR-Blunt II cloning vector and introduced into both strains by electroporation. Similarly cloning and transferring the additional wzzB genes from E.coli K-12 and Salmonella typhimurium LT 2; likewise, the fepE genes from E.coli O25K5H1, GAR2401, O25a ETEC NR-5, O157: H7: K-and Salmonella typhimurium LT2 were cloned and transferred similarly.
Bacteria were grown overnight in LB medium, LPS was extracted with phenol, resolved by SDS PAGE (4-12% acrylamide) and stained. Each well of the gel was loaded with the same number of bacterial cells (approximately 2 OD)600Unit) of LPS extracted. The size of LPS was estimated according to the internal native e.coli LPS standard and by counting the ladder bands discernible from a subset of samples showing a broad chain length distribution (differing by one repeat unit). On the left of fig. 13A, the LPS map of plasmid transformants of O25a O25K5H Δ wzzB is shown; the right is a similar pattern for the O25b GAR 2401. delta. wzzB transformant. Immunoblots of duplicate gels probed with O25-specific sera are shown in fig. 13B.
The results of this experiment show that introduction of the homologous wzzB gene into the E.coli O25a Δ wzzB host restores expression of the short O25 LPS (10-20 fold), as does Salmonella LT2 wzzB. The introduction of the O25b wzzB gene from GAR2401 was not so, indicating that the wzzB enzyme from this strain is defective. Comparison of the E.coli WzzB amino acid sequences suggests that the A210E and P253S substitutions may be responsible. Notably, salmonella LT2 fepE and escherichia coli fepE from O25a O25K5H1 conferred the ability to express Very Long (VL) OAg LPS, with salmonella LT2 fepE resulting in OAg sizes exceeding those conferred by escherichia coli fepE.
A similar expression pattern was observed for GAR2401 Δ wzzB transformants: coli O25a or K12 strain wzzB restored the ability to produce short LPS. Salmonella LT2fepE produces the longest LPS, E.coli fepE produces a slightly shorter LPS, and Salmonella LT2 wzB produces a medium size long LPS (L). In a separate experiment with transformants of E.coli O25a Δ wzB, the ability of other E.coli fepE genes to produce very long LPS was evaluated. The fepE genes from GAR2401, O25a ETEC strain, and O157 shigella toxin-producing strain also conferred the ability to produce very long LPS (but not as long as LPS produced with salmonella LT2 fepE) (fig. 14).
After determining that salmonella LT2fepE produces the longest LPS among the evaluated polymerase regulators in serotypes O25a and O25b, we next sought to determine whether it also produces very long LPS in other e. Wild-type bacteremic isolates of serotypes O1, O2, O6, O15 and O75 were transformed with the salmonella fepE plasmid and LPS extracted. The results shown in fig. 15 demonstrate that salmonella fepE is able to confer the ability to produce very long LPS in other circulating serotypes associated with blood infections. The results also indicate that plasmid-based expression of salmonella fepE appears to override the control of chain length typically imposed by endogenous wzzB in these strains.
Plasmid-based expression of O-antigens in a common E.coli host strain.
From a bioprocess development perspective, the ability to produce different serotypes of O-antigen in a common E.coli host rather than multiple strains would greatly simplify the production of a single antigen. To this end, O-antigen gene clusters from different serotypes were amplified by PCR and cloned into a low copy number plasmid (pBAD33) under the control of an arabinose regulated promoter. This plasmid is compatible (can coexist) with the salmonella LT2 fepE plasmid in e.coli, since it contains a different (p15a) replicon and a different selectable marker (chloramphenicol vs kanamycin). In a first experiment, a subclone of the pBAD 33O 25b operon plasmid was co-transfected with the Salmonella LT2 fepE plasmid into GAR 2401. delta. wzzB and transformants were grown in the presence or absence of 0.2% arabinose. The results shown in FIGS. 16A-16B demonstrate that very long O-antigen LPS was produced in an arabinose-dependent manner.
The o-antigen gene clusters cloned from other serotypes were similarly evaluated, and the results are shown in fig. 17. Co-expression of the Salmonella LT2 fepE and the pBAD33-OAg plasmid resulted in detectable long chain LPS corresponding to the O1, O2 (for two of the four clones), O16, O21 and O75 serotypes. For unknown reasons, the pBAD33-O6 plasmid failed to produce detectable LPS in all four isolates tested. Although the expression level is variable, the results indicate that it is feasible to express long-chain O-antigens in a common host. However, in some cases, further optimization (e.g., by modifying the plasmid promoter sequence) may be required to increase expression.
The profile of LPS from different serotype O25 escherichia coli strains with and without the salmonella LT2 fepE plasmid is shown in fig. 18. Fermentation, O-antigen extraction and purification of two strains were studied: GAR2831 for producing natural short O25b OAg; and GAR 2401. delta. wzzB/fepE for the production of long O25b OAg. The corresponding short and long forms of LPS shown in the SDS-PAGE gels are highlighted in red. The polysaccharides are extracted directly from the fermented bacteria with acetic acid and purified. Size exclusion chromatography profiles of purified short and long or very long O25B polysaccharide are shown in fig. 19A-19B. The properties of two batches of short polysaccharide (from GAR2831) were compared to a single very long polysaccharide preparation (from strain GAR 2401. delta. wzzB/fepE). The molecular weight of the long O-antigen is 3.3 times that of the short O-antigen and the number of repeat units is estimated to be about 65 (very long) versus about 20. See table 13.
Watch 13
Polysaccharide batch number Naturally occurring Naturally occurring Modified (long chain)
Polysaccharide batch number 709766-24A 709722-24B 709766-25A
Polysaccharide MW (kDa) 17.3 16.3 55.3
Number of repeating units 20 19 64
Very long O25b O-antigen polysaccharide was reacted with diphtheria toxoid CRM using conventional reductive amination methods197And (6) conjugation. To different extents: moderate (5.5%), low (4.4%) and high (8.3%) periodate activation three different batches of glycoconjugates were prepared. The resulting preparation and unconjugated polysaccharide were shown to be free of endotoxin contamination) (table 14).
Groups of four rabbits (female new zealand white rabbits) per group were individually inoculated with 10 meg glycoconjugate and 20 meg QS21 adjuvant and serum samples (VAC-2017-PRL-EC-0723) according to the protocol shown in fig. 20A. Notably, in evaluating bacterial glycoconjugates, a dose of 10mcg is at the lower end of the range typically given to rabbits (20-50mcg being more typical). In a separate study (VAC-2017-PRL-GB-0698), a group of rabbits were also vaccinated with unconjugated polysaccharide using the same dose (10mcg polysaccharide +20mcg Qs21 adjuvant) and the same administration protocol.
Rabbit antibody responses to three O25b glycoconjugate formulations were evaluated in a LUMINEX assay in which carboxyl beads were coated with methylated human serum albumin pre-bound to unconjugated O25b long polysaccharide. The serum samples were tested for the presence of O25b specific IgG antibodies using Phycoerythrin (PE) -labeled anti-IgG secondary antibodies. The immune response curves observed in sera from the best-responding rabbits (1 out of 4 in each group) at week 0 (pre-immunization), week 6 (post-2 dose, PD2), week 8 (post-3 dose, PD3), and week 12 (post-4 dose, PD4) are shown in fig. 21A-fig. 21C. No significant preimmune serum IgG titers were detected in any of the 12 rabbits. In contrast, O25b antigen-specific antibody responses were detected in post-vaccination sera of all three groups of rabbits, with the response tending to be slightly higher for the low-activated glycoconjugate group than for the medium-or highly-activated glycoconjugate group. Maximal responses were observed at 3 time points post-dose. One rabbit in the low activation group and one rabbit in the high activation group did not respond to vaccination (non-responders).
To evaluate CRM197Effect of Carrier protein conjugation on immunogenicity of Long O25b OAg polysaccharide the presence of antibodies in rabbit sera vaccinated with unconjugated polysaccharide was compared to CRM vaccinated with low activation197The presence of antibodies in rabbit sera of glycoconjugates was compared, fig. 22A-22F. Notably, the free polysaccharide was not immunogenic and elicited little IgG response in the immune serum versus pre-immune serum (fig. 22A). In contrast, O25b OAg-CRM was inoculated from four sera over a series of serum dilutions (from 1:100 to 1:6400)197In three PD4 sera from rabbits, a mean fluorescence intensity value (MFI) for O25b OAg-specific IgG approximately ten times higher than preimmune serum levels was observed. These results demonstrate the necessity of conjugation of the carrier protein to generate IgG antibodies against the O25b OAg polysaccharide at a dose level of 10 mcg.
Bacteria grown on TSA plates were suspended in PBS and adjusted to OD600Was 2.0 and fixed in 4% paraformaldehyde in PBS. After 1h blocking in 4% BSA/PBS, the bacteria were incubated with serial dilutions of preimmune and PD3 immune sera in 2% BSA/PBS, and labeled with a second PEF (ab) antibody detects bound IgG.
O25b OAg-CRM197The specificity of the elicited O25b antibody was confirmed in flow cytometry experiments with intact bacteria. Use of PE conjugated F (ab')2The fragment goat anti-rabbit IgG detected binding of IgG to intact cells.
As shown in fig. 23A-23C, the pre-immune rabbit antibodies were unable to bind to wild-type serotype O25b isolates GAR2831 and GAR2401 or to the K-12 e.coli strain, whereas the matched PD3 antibody stained the O25b bacteria in a concentration-dependent manner. The negative control K-12 strain lacking the ability to express OAg showed only very weak PD3 antibody binding, most likely due to the presence of exposed inner core oligosaccharide epitopes on its surface. Introduction of the salmonella fepE plasmid into the wild-type O25b isolate resulted in significantly enhanced staining, consistent with the higher density of immunogenic epitopes provided by longer OAg polysaccharides.
And (4) conclusion: the results indicate that Salmonella fepE is not only a very long determinant of O-antigen polysaccharides in Salmonella species, but it also confers a very long OAg producing capability on E.coli strains of different O-antigen serotypes. This property can be exploited to produce O-antigen vaccine polysaccharides with improved properties for bioprocess development by facilitating purification and chemical conjugation with appropriate carrier proteins, as well as by potentially enhancing immunogenicity (by forming higher molecular weight complexes).
Example 14: initial rabbit studies generated a study directed against RAC O25b OAg-CRM197The first polyclonal antibody reagent of (1) and the IgG reaction
Production of growing chain O25b polysaccharide-CRM using periodate oxidation followed by conjugation using Reductive Amination Chemistry (RAC)197Conjugate (table 14). See also table 24.
TABLE 14
Figure BDA0003622971360001601
In rabbit study 1(VAC-2017-PRL-EC-0723) (also described in example 13 above) -five (5) rabbits/group, compositions with 10ug L-, M-or H-activated RAC (+ QS21) were received according to the schedule shown in fig. 20A. The absence of immunogenicity of the unconjugated free O25b polysaccharide was observed in a follow-up rabbit study (VAC-2017-PRL-GB-0698) (see figure 25).
In rabbit study 2(VAC-2018-PRL-EC-077) -2 rabbits/group, animals received a rabbit with L-RAC (AlOH) according to the schedule shown in FIG. 20B3QS21 or without adjuvant).
Rabbits 4-1, 4-2, 5-1, 5-2, 6-1 and 6-2 received the very long unconjugated O25b polysaccharide described in example 13 and the sera were tested at week 18.
More specifically, rabbit 4-1 was administered a composition containing 50ug unconjugated O25b, 100ug AlOH3A composition of adjuvants. Rabbit 4-2 was administered a composition containing 50ug unconjugated O25b, 100ug AlOH3A composition of adjuvants. Rabbit 5-1 was administered a composition containing 50ug unconjugated O25b, 50ug QS-21 adjuvant. Rabbit 5-2 was administered a composition containing 50ug unconjugated O25b, 50ug QS-21 adjuvant. Rabbit 6-1 was administered a composition containing 50ug unconjugated O25b without adjuvant. Rabbit 6-2 was administered a composition containing 50ug unconjugated O25b without adjuvant.
Example 15: rabbit study with O25b RAC conjugate: dLIA serum dilution titer
Rabbit study 2(VAC-2018-PRL-EC-077) O25b dLIA serum dilution titer control best responding rabbits from study 1 (VAC-2017-PRL-EC-0723). For these experiments, a modified direct binding Luminex assay was performed in which polylysine conjugates of O25b long O-antigen were passively adsorbed onto Luminex carboxyl beads instead of the aforementioned methylated serum albumin long O-antigen mixture. The use of polylysine-O25 b conjugate improved the sensitivity of the assay and the quality of the IgG concentration-dependent response, allowing determination of serum dilutions by using curve fitting (four-parameter non-linear equation). The O25b IgG titers in the sera of the highest titer rabbits from the first study were compared to the O25b IgG titers in the sera of the second study rabbits in table 15.
Watch 15
Figure BDA0003622971360001611
Higher doses (50/20ug vs 10ug) in the second rabbit study did not increase IgG titers.
Two months of rest enhanced IgG response (not observed at shorter intervals).
Alum appears to enhance the IgG response in rabbits compared to QS21 or without adjuvant.
An opsonophagocytosis assay (OPA) was established using Baby Rabbit Complement (BRC) and HL60 cells (as a neutrophil source) to measure the functional immunogenicity of O-antigen glycoconjugates. A pre-frozen stock of the bacterium Escherichia coli GAR2831 was grown in Luria Broth (LB) at 37 ℃. Cells were pelleted and suspended in PBS supplemented with 20% glycerol to a concentration of 1OD 600Unit/ml, and frozen. In U-bottom tissue culture microtiter plates, pre-titrated thawed bacteria were diluted to 0.5X 10 in HBSS (Hank's Balanced salt solution) containing 1% gelatin5CFU/ml, and 10. mu.L (10)3CFU) were combined with 20 μ L serial dilutions of serum and incubated at 5% CO2In the incubator, the mixture was shaken on a BELLCO shaker at 700rpm for 30min at 37 ℃. mu.L of 2.5% complement (preimmune serum prediluted in HBG, PEL-FREEZ 31061-3) and 20. mu.L of HL-60 cells (0.75X 10)7/ml) and 40. mu.L of HBG were added to U-bottom tissue culture microplates in 5% CO2In the incubator, the mixture was shaken on a BELLCO shaker at 700rpm for 45min at 37 ℃. Subsequently, 10. mu.L of each 100. mu.L reaction was transferred to the corresponding well of a prewetted MILLIPORE MULTICENHTS Hv filter plate prepared by applying 100. mu.L water, filtering off vacuum and applying 150. mu.L of 50% LB. The filter plate was vacuum filtered and washed with 5% CO2The culture was carried out overnight in an incubator at 37 ℃. The following day, after fixing, dyeing with coomassie dye and decolorizing with decolorizing solution, use
Figure BDA0003622971360001621
The analyzer and immucocapure software count colonies. To determine the specificity of the OPA activity, immune sera were pre-incubated with 100. mu.g/mL of purified long O25b O-antigen before mixing with the other assay components in the OPA reaction. The OPA assay included control reactions without HL60 cells or complement to demonstrate the dependence of any observed killing on these components.
Matched pre-and post-vaccination serum samples from representative rabbits from both rabbit studies were evaluated in the assay and serum dilution titers were determined (table 16, fig. 26A-26B). Pre-incubation with unconjugated O25b long O-antigen polysaccharide blocked bactericidal activity, demonstrating the specificity of OPA (fig. 19C). TABLE 16OPA Titers
Rabbits 2-3 were dosed as follows: rabbit 2-3 dosing: 10/10/10/10ug RAC conjugate + Qs21, bleed after 4 th dose (PD). Rabbits 1-2 were dosed as follows: 50/20/20/20ug RAC conjugate + Al (OH)3PD4 exsanguination.
Figure BDA0003622971360001631
Example 16: o-antigen O25b IgG levels induced by unconjugated O25b long O-antigen polysaccharide and derivatized O25b RAC/DMSO long O-antigen glycoconjugate.
Groups of 10 CD-1 mice were administered 0.2 or 2.0 μ g/animal O25b RAC/DMSO long O-antigen glycoconjugates by subcutaneous injection at weeks 0, 5 and 13, and blood was drawn at week 3 (post 1 dose, PD1), week 6 (post 2 dose, PD2) and week 13 (post 3 dose, PD3) time points for immunogenicity testing. The level of antigen-specific IgG was determined by the quantitative Luminex assay (see details in example 15) using O25 b-specific mouse mAb as an internal standard. Baseline IgG levels were determined in sera pooled from 20-fold randomly selected uninoculated mice (dashed line). Free unconjugated O25b long O-antigen polysaccharide immunogen induced no IgG above baseline levels at any time point. In contrast, O25b-CRM at two doses 197IgG responses were observed after RAC long conjugate glycoconjugates: a strong consistent IgG response was observed in PD3, a moderate and moderate IgG response was observed in PD2More variable IgG levels. GMT IgG values (ng/ml) are presented as 95% CI error bars. See fig. 27A-27C.
Example 17: specificity of O25b Baby Rabbit Complement (BRC) OPA.
A-B) post-O25B RAC/DMSO long O-antigen immunization sera from rabbits 2-3 and 1-2 (but not matched pre-immune control sera) showed bactericidal OPA activity. C) OPA activity of immune sera from rabbits 1-2 was blocked by pre-incubation with 100. mu.g/mL long O-antigen O25b polysaccharide. Strain GAR2831 bacteria were incubated with HL60, 2.5% BRC and serial dilutions of serum for 1h at 37 ℃ and surviving bacteria were counted by counting filter plate microcolonies (CFU). See fig. 26A-26C.
Example 18: RAC and eTEC O25b long glycoconjugates are more immunogenic than single-ended glycoconjugates.
BRC OPA assays were performed with the carbapenem-resistant fluoroquinolone-resistant MDR strain Atlas 187913. One group of 20 CD-1 mice was inoculated with 2 μ g of glycoconjugate according to the same schedule as shown in fig. 28A-28B and OPA response was measured at time points after the 2 nd dose (PD2) (fig. 28A) and after the 3 rd dose (PD3) (fig. 28B). Bars represent GMT with 95% CI. Indicating a non-responder rate above the non-vaccinated baseline. Different sets of log transformed data were evaluated using unpaired t-test with Welch correction (Graphpad Prism) to assess whether the differences were statistically significant. The results are summarized in table 17. See fig. 28A-28B. In mice vaccinated with 2 μ g of eTEC O1a long glycoconjugate, higher OPA titers were observed against O1a, PD2 and PD3 (data not shown) than against O25b, PD2 and PD3, respectively, as shown in table 17.
TABLE 17
Figure BDA0003622971360001641
Example 19: OPA immunogenicity of eTEC chemistry can be improved by altering the level of polysaccharide activation.
BRC OPA assays were performed with the carbapenem-resistant fluoroquinolone-resistant MDR strain Atlas 187913. Groups of 20 CD-1 mice were inoculated with 0.2. mu.g or 2. mu.g of the indicated long O25b eTEC glycoconjugate and OPA responses were measured at PD2 time. Aggregated log transformed data (Aggregated log transformed data) from 4% versus 17% activation groups were evaluated using unpaired t-test with Welch correction (Graphpad Prism) to confirm that the difference in OPA response was statistically significant. GMT and responder ratios for each group are summarized in table 18. See fig. 29.
Watch 18
Description of the invention Responder% (N/N) Geometric mean titer
eTEC
4% long activation (0.2. mu.g) 35(7/20) 628
eTEC 4% long activation (0.2. mu.g) 65(13/20) 8,185
eTEC 10% long activation (0.2. mu.g) 45(9/20) 1,085
eTEC 10% long activation (0.2. mu.g) 90(18/20) 27,368
eTEC 17% long activation (0.2. mu.g) 70(14/20) 3,734
eTEC 17% long activation (0.2. mu.g) 80(16/20) 25,461
Example 20: challenge studies showed that long e.coli O25b eTEC conjugate elicited protection after three doses.
By 1x 109Bacterial IP challenge of individual strains GAR2831 groups of 20 × CD-1 mice immunized with a dose of 2 μ g according to the schedule shown. Subsequent survival was monitored for six days. Groups of mice vaccinated with 4%, 10% or 17% level activated eTEC glycoconjugate were protected from lethal infection, whereas unvaccinated control mice or mice vaccinated with 2 μ g unconjugated O25b long polysaccharide were not immunized against lethal infection. See fig. 30A-30B.
Example 21: methods of making eTEC-linked glycoconjugates
Activation of the sugar and thiolation of cystamine dihydrochloride. The sugars were reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the solution was determined by Karl Fischer (KF) analysis and adjusted to a moisture content of 0.1-1.0%, typically 0.5%.
To initiate activation, solutions of 1,1 '-carbonyl-bis-1, 2, 4-triazole (CDT) or 1, 1' -Carbonyldiimidazole (CDI) were freshly prepared in DMSO at a concentration of 100 mg/mL. The sugars were activated with varying amounts of CDT/CDI (1-10 molar equivalents) and the reaction was allowed to proceed at room temperature or 35 ℃ for 1-5 hours. Water was added to quench any residual CDI/CDT in the activation reaction solution. Calculations were made to determine the amount of water added and to allow a final water content of 2-3% of the total water content. The reaction was allowed to proceed at room temperature for 0.5 hours. Cystamine dihydrochloride was freshly prepared in anhydrous DMSO at a concentration of 50 mg/mL. The activated saccharide is reacted with 1-2 molar equivalents of cystamine dihydrochloride. Alternatively, the activated sugar is reacted with 1-2 molar equivalents of cysteamine hydrochloride. The thiolation reaction is allowed to proceed at room temperature for 5-20 hours to produce a thiolated sugar. The level of thiolation is determined by the amount of CDT/CDI added.
Reduction and purification of the activated thiolated saccharide. To the thiolated saccharide reaction mixture is added a 3-6 molar equivalent solution of tris (2-carboxyethyl) phosphine (TCEP) and allowed to proceed at room temperature for 3-5 hours. The reaction mixture was then diluted 5-10 fold by the addition of pre-cooled 10mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of the thiolated sugar was performed against a 30-40 fold osmotic volume of pre-cooled 10mM sodium dihydrogen phosphate. Aliquots of the activated thiolated sugar residues were removed to determine sugar concentration and thiol content (Ellman) measurements.
Activation and purification of bromoacetylated carrier proteins. The free amino groups of the carrier protein are bromoacetylated by reaction with a bromoacetylating agent such as bromoacetic acid N-hydroxysuccinimide ester (BAANS), bromoacetyl bromide, or another suitable reagent.
Prior to activation, the carrier protein (in 0.1M sodium phosphate, pH 8.0. + -. 0.2) is first maintained at 8. + -. 3 ℃ and at a pH of about 7. N-hydroxysuccinimide ester of bromoacetic acid (BAANS) (20mg/mL) was added to the protein solution as a stock dimethyl sulfoxide (DMSO) solution at a BAANS: protein (w/w) ratio of 0.25-0.5. The reaction was gently mixed at 5 + -3 deg.C for 30-60 minutes. For example, the resulting bromoacetylated (activated) protein is purified by ultrafiltration/diafiltration using a 10kDa MWCO membrane, using 10mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein was estimated by Lowry protein assay.
The degree of activation was determined by total bromide determination using ion exchange liquid chromatography in combination with suppressed conductivity detection (ion chromatography). The bound bromide on the activated bromoacetylated protein is cleaved from the protein in the preparation of the assay sample and quantified along with any free bromide that may be present. Any remaining covalently bound bromine on the protein is converted to ionic bromide and released by heating the sample in alkaline 2-mercaptoethanol.
Bromoacetylated CRM197Activation and purification. CRM with 10mM phosphate buffered 0.9% NaCl pH 7(PBS)197Diluting to 5mgmL, then made up with 1M stock solution to 0.1M NaHCO3The pH was 7.0. Stock solutions of BAANS using 20mg/mL DMSO in 1:0.35(w: w) CRM197BAANS proportion BAANS is added. The reaction mixture was incubated between 3 ℃ and 11 ℃ for 30min to 1 hour, then purified by ultrafiltration/diafiltration using a 10K MWCO membrane and 10mM sodium phosphate/0.9% NaCl, pH 7.0. Purified activated CRM as determined by Lowry assay197To determine the protein concentration, it was then diluted to 5mg/mL with PBS. Sucrose was added as a cryoprotectant at 5% wt/vol and the activated protein was frozen and stored at-25 ℃ until conjugation was required.
CRM197The bromoacetylation of lysine residues of (a) is very consistent, resulting in activation of 15 to 25 lysines from 39 available lysines. The reaction produces high yields of active protein.
Conjugation of activated thiolated saccharides to bromoacetylated carrier proteins. Followed by the addition of bromoacetylated carrier protein and activated thiolated saccharide. The sugar/protein input ratio was 0.8. + -. 0.2. The reaction pH was adjusted to 9.0. + -. 0.1 with 1M NaOH solution. The binding reaction was allowed to proceed for 20. + -. 4 hours at 5 ℃.
Capping of residual reactive functional groups. The unreacted bromoacetylated residues on the carrier protein are quenched by reaction with 2 molar equivalents of N-acetyl-L-cysteine as a capping agent for 3-5 hours at 5 ℃. The remaining free thiol groups were blocked with 4 molar equivalents of Iodoacetamide (IAA) at 5 ℃ for 20-24 hours.
Purification of eTEC-linked glycoconjugates. The conjugation reaction (after IAA capping) mixture was filtered through a 0.45 μm filter. Ultrafiltration/diafiltration of the glycoconjugate was performed with 5mM succinate-0.9% saline, pH 6.0. The glycoconjugate residue was then filtered through a 0.2 μm filter. Aliquots of the glycoconjugates were removed for assay. The remaining glycoconjugates were stored at 5 ℃. See tables 21, 22, 23, 24 and 25.
Example 22: preparation of E.coli-O25B ETEC conjugate
Activation process-activation of lipopolysaccharide of Escherichia coli-O25 b. The lyophilized E.coli O25b polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized O25b/DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the O25b/DMSO solution to bring the moisture content to 0.5%.
To initiate activation, 100mg/mL of 1, 1' -Carbonyldiimidazole (CDI) was freshly prepared in DMSO solution. coli-O25 b polysaccharide was activated with varying amounts of CDI prior to the thiolation step. CDI activation is carried out at room temperature or 35 ℃ for 1-3 hours. Water was added to quench any residual CDI in the activation reaction solution. Calculations were made to determine the amount of water added and to allow a final water content of 2-3% of the total water content. The reaction was allowed to proceed at room temperature for 0.5 hours.
Thiolation of the activated E.coli O25b polysaccharide. Cysteamine dihydrochloride is freshly prepared in anhydrous DMSO and 1-2 molar equivalents of cysteamine dihydrochloride is added to the activated polysaccharide reaction solution. The reaction was allowed to proceed for 20. + -. 4 hours at room temperature.
Reduction and purification of activated thiolated E.coli-O25 b polysaccharide. To the thiolated sugar reaction mixture is added a 3-6 molar equivalent solution of tris (2-carboxyethyl) phosphine (TCEP) and allowed to proceed at room temperature for 3-5 hours. The reaction mixture was then diluted 5-10 fold by the addition of pre-cooled 10mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of thiolated sugar using 5K MWCO ultrafiltration membrane cartridges against 40 osmotic volumes of pre-cooled 10mM sodium dihydrogen phosphate. The thiolated O25b polysaccharide residue was removed for sugar concentration and thiol (Ellman) measurements. A flow chart of the activation process is provided in fig. 32A.
Conjugation Process-thiolated E.coli-O25 b polysaccharide with Bromoacetylated CRM197The conjugation of (2). CRM was activated alone by bromoacetylation as described in example 21197A carrier protein, which is then reacted with activated E.coli-O25 b polysaccharide to effect a conjugation reaction. Acetylating bromine into CRM197And the thiolated O25b polysaccharide were mixed together in a reaction vessel. The sugar/protein input ratio was 0.8. + -. 0.2. The reaction pH was adjusted to 8.0-10.0. The conjugation reaction was allowed to proceed for 20. + -. 4 hours at 5 ℃.
Bromoacetylated CRM197And thiolated E.coli-O25 b polysaccharideEnd capping of the reactive group. By combining CRM197Unreacted bromoacetylated residues on proteins are reacted with 2 molar equivalents of N-acetyl-L-cysteine at 5 ℃ for 3-5 hours followed by capping any remaining free thiol groups of the thiolated O25 b-polysaccharide with 4 molar equivalents of Iodoacetamide (IAA) at 5 ℃ for 20-24 hours197And (4) blocking unreacted bromoacetylation residues on the protein.
Purification of eTEC-linked e.coli-O25 b glycoconjugate. The conjugation solution was filtered through a 0.45 μm or 5 μm filter. Diafiltration of the O25b glycoconjugate was performed using 100K MWCO ultrafiltration membrane cartridges. Diafiltration was performed against 5mM succinate-0.9% saline, pH 6.0. The E.coli-O25 b glycoconjugate 100K residue was then filtered through a 0.22 μm filter and stored at 5 ℃.
A flow chart of the conjugation process is provided in fig. 32B.
As a result, the
The reaction parameters and characterization data for several batches of E.coli-O25 b eTEC glycoconjugate are shown in Table 19. CDI activation-thiolation with cystamine dihydrochloride yielded glycoconjugates with sugar yields of 41 to 92% and free sugars <5 to 14%. See also tables 21, 22, 23, 24 and 25.
TABLE 19 Experimental parameters and characterization data for E.coli-O25 b eTEC conjugates
Figure BDA0003622971360001691
Example 23: escherichia coli O-antigen polysaccharide-CRM197Method for preparing eTEC conjugate (O-antigen suitable for E.coli serotypes O25b, O1a, O2 and O6)
And (4) activating the polysaccharide.
Coli O-antigen polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). To initiate activation, various amounts of 1, 1' -Carbonyldiimidazole (CDI) (1-10 molar equivalents) were added to the polysaccharide solution and the reaction was allowed to proceed at room temperature or 35 ℃ for 1-5 hours. Then, water (2-3%, v/v) was added to quench any residual CDI in the activation reaction solution. After allowing the reaction to proceed at room temperature for 0.5 hours, 1-2 molar equivalents of cystamine dihydrochloride was added. The reaction is allowed to proceed at room temperature for 5-20 hours and then treated with 3-6 molar equivalents of tris (2-carboxyethyl) phosphine (TCEP) to yield the thiolated saccharide. The level of thiolation is determined by the amount of CDI added.
The reaction mixture was then diluted 5-10 fold by the addition of pre-cooled 10mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of thiolated sugar was performed on 30-40 dialysis volumes of pre-cooled 10mM sodium dihydrogen phosphate. An aliquot of the activated thiolated sugar residue was removed to determine the sugar concentration and thiol content (Ellman) measurements.
Carrier protein (CRM)197) Activation of
CRM was first applied prior to activation197(in 0.1M sodium phosphate, pH 8.0. + -. 0.2) at 8. + -. 3 ℃ and a pH of about 8. N-hydroxysuccinimide ester of bromoacetic acid (BAANS) (20mg/mL) was added to the protein solution as a stock dimethyl sulfoxide (DMSO) solution at a BAANS: protein (w/w) ratio of 0.25-0.5. The reaction was gently mixed for 30-60 minutes at 5. + -. 3 ℃. For example, the resulting bromoacetylated (activated) protein is purified by ultrafiltration/diafiltration using a 10kDa MWCO membrane, using 10mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein was estimated by Lowry protein assay.
Conjugation
Followed by activated CRM197And activated E.coli O-antigen polysaccharide were added to the reactor and mixed. The sugar/protein input ratio was 1. + -. 0.2. The reaction pH was adjusted to 9.0. + -. 0.1 with 1M NaOH solution. The binding reaction was allowed to proceed for 20. + -. 4 hours at 5 ℃. The unreacted bromoacetylated residues on the carrier protein are quenched by reaction with 2 molar equivalents of N-acetyl-L-cysteine as a capping agent for 3-5 hours at 5 ℃. The remaining free thiol groups were blocked with 4 molar equivalents of Iodoacetamide (IAA) at 5 ℃ for 20-24 hours. The reaction mixture was then purified using ultrafiltration/diafiltration against 5mM succinate-0.9% saline, pH 6.0. The purified conjugate was then filtered through a 0.2 μm filter. See tables 21, 22, 23 Table 24 and table 25.
Example 24: general procedure-conjugation of O-antigens (from E.coli serotypes O1, O2, O6, 25b) polysaccharides by Reductive Amination Chemistry (RAC)
Conjugation in dimethyl sulfoxide (RAC/DMSO)
Activated polysaccharides
Polysaccharide oxidation was performed in 100mM sodium phosphate buffer (pH 6.0. + -. 0.2) by adding calculated amounts of 500mM sodium phosphate buffer (pH 6.0) and water for injection (WFI) in sequence to give a final polysaccharide concentration of 2.0 g/L. The reaction pH was adjusted to about pH 6.0 if necessary. After pH adjustment, the reaction temperature was cooled to 4 ℃. The oxidation is initiated by the addition of about 0.09 to 0.13 molar equivalents of sodium periodate. The oxidation reaction was carried out at 5. + -. 3 ℃ for about 20. + -. 4 hours.
The activated polysaccharide was concentrated and diafiltered using a 5K MWCO ultrafiltration cassette. Diafiltration was performed against 20 diafiltration volumes of WFI. The purified activated polysaccharide was then stored at 5 ± 3 ℃. The purified activated sugars are characterized inter alia in that: (i) sugar concentration measured by colorimetric assay; (ii) aldehyde concentration measured by colorimetric assay; (iii) the degree of oxidation; and (iv) molecular weight as measured by SEC-MALLS.
Compounding the activated polysaccharide with sucrose excipient, and freeze drying
The activated polysaccharide was mixed with sucrose at a ratio of 25 grams of sucrose per gram of activated polysaccharide. The vial of complex mixture was then lyophilized. After lyophilization, the vials containing the lyophilized activated polysaccharide were stored at-20 ± 5 ℃. CRM to be calculated197Protein shells were frozen (shell-FROZEN) and lyophilized separately. Storage of lyophilized CRM at-20 + -5 deg.C197。
Reconstituting the lyophilized activated polysaccharide and carrier protein
The lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). After complete dissolution of the polysaccharide, the lyophilized CRM was added197Equal amount of anhydrous DMSO was added for reconstitution.
Conjugation and end capping
Reconstituted activated polysaccharide with reconstituted CRM in a reaction vessel197Combination ofFollowed by thorough mixing to obtain a clear solution, and then initial conjugation with sodium cyanoborohydride. The final polysaccharide concentration in the reaction solution was about 1 g/L. Conjugation was initiated by adding 0.5-2.0 Meq of sodium cyanoborohydride to the reaction mixture and incubating at 23 ± 2 ℃ for 20-48 hours. By adding 2MEq of sodium borohydride (NaBH)4) The conjugation reaction is terminated by capping the unreacted aldehyde. The capping reaction was continued at 23. + -. 2 ℃ for 3. + -. 1 h.
Purification of conjugates
The conjugate solution was diluted 1:10 with cooled 5mM succinate-0.9% saline (pH 6.0) in preparation for purification by tangential flow filtration using a 100-K MWCO membrane. The diluted conjugate solution was passed through a 5 μm filter and diafiltered using 5mM succinate/0.9% saline (pH 6.0) as the medium. After diafiltration was complete, the conjugate residue was transferred through a 0.22 μm filter. The conjugate was further diluted with 5mM succinate/0.9% saline (pH 6) to reach a target sugar concentration of approximately 0.5 mg/mL. Alternatively, the conjugate was purified by tangential flow filtration using a 100-K MWCO membrane using 20mM histidine-0.9% saline (pH 6.5). The final 0.22 μm filtration step was completed to obtain the immunogenic conjugate. See tables 21, 22, 23, 24 and 25.
Example 25: conjugation in aqueous buffer (RAC/aqueous) suitable for E.coli serotypes O25B, O1A, O2 and O6
Polysaccharide activation and diafiltration were performed in the same way as DMSO-based conjugation.
Filtering the activated saccharide to CRM at a polysaccharide to protein mass ratio in the range of 0.4 to 2w/w, depending on serotype197And (4) mixing. The input ratio is selected to control polysaccharide to CRM in the resulting conjugate197The ratio of (a) to (b).
The mixed mixture was then lyophilized. After conjugation, the polysaccharide and protein mixture was dissolved in 0.1M sodium phosphate buffer, with a polysaccharide concentration ranging from 5 to 25g/L depending on the serotype, and a pH adjusted between 6.0 and 8.0 depending on the serotype. Adding 0.5-2.0 Meq of sodium cyanoborohydride into the reaction mixture, and incubating at 23 +/-2 ℃ for 20-48 hoursTo initiate conjugation. By adding 1-2MEq of sodium borohydride (NaBH)4) The conjugation reaction is terminated by capping the unreacted aldehyde.
Alternatively, the filtered activated saccharide and a calculated amount of CRM197The proteins were frozen and lyophilized separately, then combined after dissolution in 0.1M sodium phosphate buffer, and then subsequent conjugation can be performed as described above.
Table 20 summarizes the results from conjugates prepared in DMSO and aqueous buffer
RAC/DMSO RAC/Water-based
Polysaccharides MW (kDa) 48K 46K
Degree of Oxidation (DO) 12 12
Sugar/protein ratio 0.8 1.0
Free sugar% <5％ 32％
Conjugate MW, kDa by SEC-MALLS 7950 260
Example 26: escherichia coli O-antigen polysaccharide-CRM197Process for the preparation of single-ended conjugates
Lipopolysaccharide (LPS) is a common component of the outer membrane of gram-negative bacteria, including lipid a, the core region, and the O-antigen (also known as O-specific polysaccharide or O-polysaccharide). The O-antigen repeat units of different serotypes differ in their composition, structure and serological characteristics. The O-antigen used in the present invention is attached to a core domain containing a sugar unit called 2-keto-3-deoxyoctanoic acid (KDO) at its chain end. Unlike some conjugation methods based on random activation of polysaccharide chains (e.g. activation with sodium periodate or carbodiimide). The present invention discloses a conjugation process comprising selective activation of KDO with a dithioamine linker after exposure of the thiol functionality, followed by its reaction with bromine-activated CRM197Protein conjugation, as shown in figure 31 (preparation of single-ended conjugates).
Conjugation based on cystamine linker (A1)
The O-antigen polysaccharide and cystamine (50-250 molar equivalents of KDO) were mixed in phosphate buffer and the pH was adjusted to 6.0-7.0. To the mixture was added sodium cyanoborohydride (NaCNBH) 3) (5-30 molar equivalents of KDO) and the mixture is stirred at 37 ℃ for 48-72 hours. After cooling to room temperature and dilution with an equal volume of phosphate buffer, the mixture was treated with tris (2-carboxyethyl) phosphine (TCEP) (1.2 molar equivalents of cystamine were added). The mixture was then purified by diafiltration against a 10mM sodium dihydrogen phosphate solution using a 5KDa MWCO membrane to provide the thiol-containing O-antigen polysaccharide. The mercaptan content can be determined by an Ellman assay.
Then by reacting the thiol-activated O-antigen polysaccharide with bromine-activated CRM197The proteins were mixed in a ratio of 0.5-2.0 for conjugation. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was carried out at 5 ℃ for 24. + -. 4 hours. By reacting unreacted bromine residues on the carrier protein with2 molar equivalents of N-acetyl-L-cysteine at 5 ℃ for 3-5 hours to quench the unreacted bromine residues. Then 3 molar equivalents of iodoacetamide (in relation to the added N-acetyl-L-cysteine) were added to cap the remaining free thiol groups. The capping reaction was carried out at 5 ℃ for an additional 3-5 hours and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/diafiltration using a 30KDa MWCO membrane against 5mM succinate-0.9% saline (pH 6.0). See tables 21, 22, 23, 24 and 25.
Example 27: conjugation based on a 3, 3' -dithiobis (malonic dihydrazide) linker (A4)
The O-antigen polysaccharide and 3, 3' -dithiobis (malonic dihydrazide) (5-50 molar equivalents of KDO) were mixed in acetate buffer, and the pH was adjusted to 4.5-5.5. To the mixture was added sodium cyanoborohydride (NaCNBH)3) (5-30 molar equivalents of KDO) and the mixture is stirred at 23-37 ℃ for 24-72 hours. The mixture was then treated with tris (2-carboxyethyl) phosphine (TCEP) (1.2 molar equivalents of 3, 3' -dithiobis (malonic acid dihydrazide) linker were added). The mixture was then purified by diafiltration against a 10mM sodium dihydrogen phosphate solution using a 5KDa MWCO membrane to provide the thiol-containing O-antigen polysaccharide. The mercaptan content can be determined by an Ellman assay.
Then by reacting the thiol-activated O-antigen polysaccharide with bromine-activated CRM197The proteins were mixed in a ratio of 0.5-2.0 for conjugation. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was carried out at 5 ℃ for 24. + -. 4 hours. Quenching the unreacted bromine residues on the carrier protein by reacting them with 2 molar equivalents of N-acetyl-L-cysteine at 5 ℃ for 3-5 hours. Then 3 molar equivalents of iodoacetamide (in relation to the added N-acetyl-L-cysteine) were added to cap the remaining free thiol groups. The capping reaction was carried out at 5 ℃ for an additional 3-5 hours and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/diafiltration using a 30KDa MWCO membrane against 5mM succinate-0.9% saline (pH 6.0).
Example 28: conjugation based on 2,2 '-dithio-N, N' -bis (ethane-2, 1-diyl) bis (2- (aminooxy) acetamide) linker (A6)
The O-antigen polysaccharide was mixed with 2,2 '-dithio-N, N' -bis (ethane-2, 1-diyl) bis (2- (aminooxy) acetamide) (5-50 molar equivalents of KDO) in acetate buffer, and the pH was adjusted to 4.5-5.5. The mixture was then stirred at 23-37 deg.C for 24-72 hours, followed by the addition of sodium cyanoborohydride (NaCNBH)3) (5-30 molar equivalents of KDO) and the mixture is stirred for a further 3-24 hours. The mixture was then treated with tris (2-carboxyethyl) phosphine (TCEP) (1.2 molar equivalents of linker added). The mixture was then purified by diafiltration against a 10mM sodium dihydrogen phosphate solution using a 5KDa MWCO membrane to provide the thiol-containing O-antigen polysaccharide. The mercaptan content can be determined by an Ellman assay.
Then by reacting the thiol-activated O-antigen polysaccharide with bromine-activated CRM197The proteins were mixed in a ratio of 0.5-2.0 for conjugation. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was carried out at 5 ℃ for 24. + -. 4 hours. Quenching the unreacted bromo residue on the carrier protein by reacting it with 2 molar equivalents of N-acetyl-L-cysteine at 5 ℃ for 3-5 hours. Then 3 molar equivalents of iodoacetamide (in relation to the added N-acetyl-L-cysteine) were added to cap the remaining free thiol groups. The capping reaction was carried out at 5 ℃ for an additional 3-5 hours and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/diafiltration using a 30KDa MWCO membrane against 5mM succinate-0.9% saline (pH 6.0).
Example 29: bromine activated CRM197Preparation of
CRM197Prepared in a 0.1M sodium phosphate (pH 8.0 + -0.2) solution and cooled to 5 + -3 deg.C. N-hydroxysuccinimide ester of bromoacetic acid (BAANS) (20mg/mL) was added to the protein solution as a stock dimethyl sulfoxide (DMSO) solution at a BAANS: protein (w/w) ratio of 0.25-0.5. The reaction was gently mixed for 30-60 minutes at 5. + -. 3 ℃. For example, ultrafiltration using 10mM phosphate (pH 7.0) buffer, by using a 10kDa MWCO membraneThe resulting bromoacetylated (activated) protein was purified by filtration/diafiltration. After purification, the protein concentration of the bromoacetylated carrier protein was estimated by Lowry protein assay.
TABLE 21O 1a conjugates
Figure BDA0003622971360001751
Figure BDA0003622971360001761
TABLE 22O2 conjugates
Figure BDA0003622971360001762
TABLE 23O6 conjugates
Figure BDA0003622971360001763
Figure BDA0003622971360001771
TABLE 24O25b conjugates
Figure BDA0003622971360001772
TABLE 25O25b K-12 conjugates
Figure BDA0003622971360001781
Example 29: preparation of Escherichia coli O-Ag-TT conjugate
50mg of lyophilized E.coli serotype O25b long polysaccharide (batch No. 709766-30) (about 6.92mg/mL, MW: about 39kDa) was used for Tetanus Toxoid (TT) conjugation.
The E.coli serotype O1a long polysaccharide 710958-3 (about 6.3mg/mL, MW: about 44.3kDa) (50mg,7.94mL) was lyophilized.
The E.coli serotype O6 long polysaccharide 710758-1 (about 16.8mg/mL, MW: about 44kDa) (50mg,2.98mL) was lyophilized.
Each of the lyophilized polysaccharides listed above was dissolved in WFI to a concentration of approximately 5-10mg/mL, 0.5mL (100mg (1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) solution in 1mL acetonitrile) was added and stirred at room temperature Triethylamine (TEA)0.2M (2mL) was added and stirred at room temperature.
Preparation of Tetanus Toxoid (TT): TT (100mg,47mL) was concentrated to about 20mL, washed twice with brine (2 × 50mL) using filter tube. It was then diluted with HEPES and saline to a final HEPES concentration of approximately 0.25M.
TT was prepared as described above and the pH of the reaction was adjusted to about 9.1-9.2. The reaction mixture was stirred at room temperature
After 20-24 hours, the reaction was quenched with glycine (0.5 mL). Thereafter, it was concentrated using MWCO regenerated cellulose membrane and the brine was diafiltered. And (4) filtering and analyzing. See table 26.
Table 26 exemplary embodiments:
Figure BDA0003622971360001791
example 30: other results of O-antigen fermentation, purification and conjugation
The exemplary methods described below are generally applicable to all E.coli serotypes. Production of each polysaccharide involves batch fermentation followed by chemical inactivation prior to downstream purification.
And (4) strain and storage. The strain used for the biosynthesis of the short-chain O-antigen is the clinical wild-type strain of Escherichia coli. Long-chain O-antigens are produced using derivatives of short-chain producers which have been engineered by the Wanner-Datsenko method, have a deletion of the native wzzzb gene, and are complemented by a "long-chain" extender function fepE from Salmonella. The fepE function is expressed from its native promoter on either a high copy "topo" colE 1-based vector or a low copy derivative of the colE 1-based vector pET30a from which the T7 promoter region has been deleted.
By culturing the cells in animal-free LB or minimal medium to an OD of at least 3.0600To prepare a cell bank. The broth was then diluted in fresh medium and mixed with 80% glycerol to obtain a broth with 2.0OD600Final concentration of 20% glycerol per mL.
Culture medium for seed culture and fermentation. The seeds and fermentation medium used share the following formulation: KH (Perkin Elmer)2PO4、K2HPO4、(NH4)2SO4Sodium citrate, Na2SO4Aspartic acid, glucose, MgSO4、FeSO4-7H2O、Na2MoO4-2H2O、H3BO3、CoCl2-6H2O、CuCl2-2H2O、MnCl2-4H2O、ZnCl2And CaCl2-2H2O。
Seeds and fermentation conditions. Seeds were inoculated at 0.1% from a single seed vial. Seed flasks (seed flash) were incubated at 37 ℃ for 16-18 hours, typically to 10-20OD600/mL。
The fermentation was carried out in a 10L stainless steel in situ steam fermentor.
Inoculation of the fermenter is usually from 10OD600The first 1: 1000. The batch phase, i.e. the period during which growth is carried out on 10g/L of glucose batch, generally lasts 8 hours. When glucose is depleted, the dissolved oxygen rises suddenly, at which time glucose is added to the fermentation. Then fermentation is usually carried out for 16-18 hours, the yield>120OD600/mL。
Preliminary assessment of short/long chain O-antigen production for serotypes O1a, O2, O6, and O25 b. The wild-type strains of O1a, O2, O6 and O25b were fermented to OD in batch mode in supplemented minimal medium60015-20. Upon depletion of glucose, resulting in a sudden decrease in oxygen consumption, a growth-limiting glucose feed was applied from the glucose solution for 16-18 hours. Up to 124-145OD 600Cell density in units/mL. The pH of the harvest broth was then adjusted to about 3.8 and heated to 95 ℃ for 2 hours. The hydrolyzed broth was then cooled to 25 ℃, adjusted to ph6.0, and centrifuged to remove solids. The resulting supernatant was then applied to a SEC-HPLC column for O-antigen quantification. Productivity in the range of 2240-4180mg/L was obtained. The molecular weight of the purified short chain O-antigen from these batches was found to be in the range of 10-15 kDa. It was also noted that SEC chromatograms of O2 and O6 hydrolysates showed distinct and separable mixed polysaccharides, which were not evident in O1a and O25b hydrolysates.
The long-chain forms of O1a, O2, O6, and O25b O-antigen were obtained by fermentation of wzzb-deleted versions of each strain carrying the heterologous, complementary fepE gene on a high copy kanamycin-selective topo plasmid. Fermentation was performed as for the short chain, despite the kanamycin selection. At 124-177OD600The final cell density observed at/mL correlated with O-antigen productivity of 3500-9850 mg/L. The complementation-based synthesis of long-chain O-antigens is at least as productive as the parent short-chain strain, and in some cases even more. The molecular weight of the purified O-antigen polysaccharide is 33-49kDa, or about 3 times the size of the corresponding short chain.
It was noted that the long chain hydrolysates of O2 and O6 showed evidence of mixed polysaccharide peaks, which in the case of long chain antigens were observed as shoulders on the main O-antigen peak; o1 and O25b showed no evidence of producing mixed polysaccharides, as previously seen in the short chain parent.
Growth rate inhibition was found to be associated with the presence of the topo replicon lacking fepE. In addition, Δ wzzb itself had no adverse effect on growth rate, indicating that the disturbed growth rate was delivered by the plasmid vector.
Evaluation of O11, O13, O16, O21 and O75O-antigen production of the strains. The tendency of various wild-type strains of serotypes O11, O13, O16, O21 and O75 to produce undesirable polysaccharides in fermentation was assessed by SEC-HPLC. Strains of O11, O13, O16, O21 and O75 were selected for the absence of mixed polysaccharides, and their ability to produce >1000mg/L O-antigen, and the display of antibiotic susceptibility profiles allowed for Wanner-Datsenko recombination engineering for the introduction of the Δ wzzb trait.
Chloramphenicol alternative forms of fepE and pET-fepE were constructed which allowed the introduction of fepE into O11, O13, O16, O21, and O75 wzbb strains that were commonly found to be resistant to kanamycin. The strains resulting from the fermentation carrying topo-fepE and pET-fepE were selected with chloramphenicol and the supernatant of the acid hydrolyzed broth was evaluated by SEC-HPLC. Both high copy (topo) and low copy (pET) fepE constructs direct the synthesis of O-antigen, with respective productivities comparable to the parental wild-type. No potential interference with polysaccharide expression was observed.
Evaluation of growth rates of strains carrying the wzzb plasmid showed that O11, O13, and O21 were retarded by the presence of topo-fepE, but not by pET-fepE; strains O16 and O75 showed acceptable growth rates regardless of the replicon chosen.
Watch 27
Figure BDA0003622971360001821
Purification of the polysaccharide involves acid hydrolysis to release the O-antigen. A crude suspension of serotype-specific E.coli culture in a fermentation reactor was directly treated with acetic acid to a final pH of 3.5. + -. 0.5 and the acidified broth was heated to a temperature of 95. + -. 5 ℃ for at least 1 hour. This treatment breaks the labile linkage between KDO at the proximal end of the oligosaccharide and lipid a, releasing the O-Ag chain. The acidified broth containing the liberated O-Ag is cooled to 20. + -. 10 ℃ and then treated with NH4OH was neutralized to pH 7. + -. 1.0. The process also includes several centrifugation, filtration and concentration/diafiltration steps.
Watch 28
Figure BDA0003622971360001831
Figure BDA0003622971360001841
Example 31: conjugation (RAC/DMSO) to O-antigens (O4, O11, O21, O75) was investigated
TABLE 29O4 conjugates
Figure BDA0003622971360001842
Figure BDA0003622971360001851
TABLE 30O11 conjugates
Figure BDA0003622971360001852
TABLE 31O21 conjugates
Figure BDA0003622971360001861
TABLE 32O75 conjugates
Figure BDA0003622971360001862
Figure BDA0003622971360001871
Example 32: prepared PLL conjugates
Watch 33
Figure BDA0003622971360001872
Example 33: stable mammalian cell expression of E.coli polypeptides Stable CHO clones expressing FimH GSD or FimH LD were generated using an SSI (site specific integration) stable expression system.
The host CHO cell is an engineered cell line from CHOK1SV GS-KO background (see, e.g., U.S. patent application 20200002727 for a description of CHOK1SV GS-KO host cell line). Briefly, a landing pad (plating pad) with the Green Fluorescent Protein (GFP) gene surrounded by two FRT sites was targeted to a transcriptional hot spot in the host cell genome. The GFP gene can be swapped with the GS gene and the target gene, which are also surrounded by FRT sites from the LVEC vector co-expressed with flippase recombinase (FLPe). This system not only has a favorable growth and productivity profile compared to random integration, but also shows genotypic and phenotypic stability for at least 100 generations.
As used herein, the term "FRT site" refers to a nucleotide sequence at which the Flippase (FLP) gene product FLP recombinase of a yeast 2 μm plasmid is capable of catalyzing site-specific recombination. Various FRT sites are known in the art. The sequences of the various FRT sites are similar in that they all contain the same 13 base pair inverted repeat flanking the 8 base pair asymmetric core region where recombination occurs. It is the asymmetric core region that determines the orientation of the sites and the variation between different FRT sites. Illustrative (non-limiting) examples of these FRT sites include naturally occurring FRT (F), and several mutant or variant FRT sites, such as FRT F1 and FRT F2.
As used herein, the term "landing pad" refers to a nucleic acid sequence comprising a first recombination target site integrated chromosomally into a host cell. In some embodiments, the landing site comprises two or more recombination target sites that are chromosomally integrated into the host cell. In some embodiments, the cell comprises 1, 2, 3, 4, 5, 6, 7, or 8 landing pads. In some embodiments, the cell comprises 1, 2, or 3 landing pads. In some embodiments, the cell comprises 4 landing pads. In some embodiments, the landing pad is integrated in up to 1, 2, 3, 4, 5, 6, 7, or 8 different chromosomal loci. In some embodiments, the landing pad is integrated in up to 1, 2, or 3 different chromosomal loci. In some embodiments, the landing pad is integrated in 4 different chromosomal loci.
The LVEC expression vector and the FLPe expression vector for FimH GSD or FimH LD were co-transfected into SSI host cells by electroporation using a BioRad Gene Pulser Xcell or Amaxa 4D-Nucleofector. The cells were then cultured in glutamine-free medium to select for cells that integrated the GS gene at the landing pad site. Cells usually recover within 2-3 weeks. Single cell cloning was then performed in 96-well plates by FACS or limiting dilution. Titers from wells with cells were fractionated to narrow to the first 48 clones. A second round of fed-batch screening was performed in 24 deep-well plates to narrow the clones to the first 12. A third round of fed-batch screening was performed in Ambr15 to narrow the clones to the first 3. Ambr250 experiments were used to identify the best clones. After the top clone was identified, its master and working cell banks were generated.
Example 34: cell line development and FimH-DSG WT and FimHLDProduction reactor expression of WT protein
The examples described herein describe the production of FimH-DSG WT and FimH from a stable CHO cell lineLDExemplary production of Wt proteins, wherein the coding sequence for each protein has been stably integrated into the CHO genome.
In a production bioreactor setting, the stable CHO cell line selected can be approximately 1 gram per liter of culture (for FimH-DSG Wt) and 250 mg per liter of culture (for FimH)LDWt) produces the target protein. Seed culture for production reactor (seed train) was continuously scaled up from vial thawing of working cell bank and used 0.3x10 in shake flask6The seeded viable cell density of one cell/ml was expanded through three passage cycles in shake flasks to provide sufficient cells for the production reactor. The cells were incubated at 36.5 ℃ with 5% CO2Growing for 3-4 days.
Production reactor was inoculated from the final shake flask with a target inoculum cell density of 1X106Individual cells/ml. A pH of 7.05(+/-0.15) and a target CO of 5-10% were used2Saturation, the production reactor was grown at 36.5 ℃ for 7 days. pH control of alkalinity, CO, by sodium/potassium bicarbonate2Spraying to control acidity. Using pure oxygen by injection Dissolved oxygen was controlled at the 40% set point. On the seventh day, the temperature was adjusted to 31 ℃. The reactor was fed on day 1 using a feed strategy that added feed related to viable cell density, which was achieved by using a feed factor of 0.75 to ensure that the feed components were not depleted during the run. The feed was then added continuously to provide the desired volume of feed over the course of one day.
The production reactor was harvested on day 13, and the harvested culture was centrifuged and subjected to 0.22 μm filtration prior to downstream processing.
The following clauses describe additional embodiments of the invention:
C1. a composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide comprising a structure selected from any one of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O, formula I, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D1, formula O63, formula O64, formula O65 (for example formula O65 (73-1 strain)), formula O65, formula O685103, formula O114, formula O685106, formula O65, formula O685106, formula O65, formula O685120, formula O103, formula O65, formula O103, formula O685106, formula O65, formula O103, formula O65, formula O685106, formula O65, formula O65, formula O103, formula O65, formula O685125, formula O65, formula O65, formula O103, formula O65, formula O65, formula O103, formula O65, formula O103, formula O65, formula O685125, formula O65, formula O103, formula O103, formula O103, formula O65, formula O103, formula O65, formula O103, formula O103, formula O65, formula O104, formula O103, formula O formula, Formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187, wherein n is an integer from 1 to 100.
C2. The composition of clause C1, wherein the sugar comprises a structure selected from the group consisting of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (180/C strain)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O (for example, formula O and formula O45rel), formula O (for example, formula O73-1 strain)), formula O, formula 143, formula O124, formula O, formula 141, formula O, formula 143, formula O, formula 141, formula 143, formula 141, formula I, Formula O147, formula O149, formula O152, formula O157, formula O158, formula O159, formula O164, formula O173, formula 62D1, formula O22, formula O35, formula O65, formula O66, formula O83, formula O91, formula O105, formula O116, formula O117, formula O139, formula O153, formula O167 and formula O172, wherein n is an integer from 20 to 100.
C3. The composition of clause C2, wherein the sugar comprises a structure selected from the group consisting of: formula O (for example, formula O1 and formula O1), formula O (for example, formula O: K and formula O: K), formula O (for example, formula O5 and formula O5 (strain 180/C)), formula O (for example, formula O: K; formula K and formula O: K), formula O (for example, formula O18A, formula O18 and formula O18B), formula O (for example, formula O23), formula O (for example, formula O25 and formula O25), formula O (for example, formula O and formula O45rel), formula O (for example, formula O73-1), formula O119, formula O114, formula O124, formula O141, formula O136, formula O, formula 143, formula O136, formula O, formula 143, formula O136, formula O136, formula I, Formula O147, formula O149, formula O152, formula O157, formula O158, formula O159, formula O164, formula O173, and formula 62D1, wherein n is an integer from 20 to 100.
C4. The composition of clause C2, comprising a structure selected from the group consisting of: formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O6 (e.g., formula O6: K2; K13; K15, and formula O6: K54), formula O15, formula O16, formula O21, formula O25 (e.g., formula O25a and formula O25b), and formula O75.
C5. The composition of clause C2, comprising a structure selected from formula O4, formula O11, formula O21, and formula O75.
C6. The composition of clause C1, wherein the saccharide does not comprise a structure selected from the group consisting of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.
C7. The composition of clause C1, wherein the saccharide does not comprise a structure selected from the group consisting of formula O12.
C8. The composition of clause C4, wherein the sugar is produced by expressing a wzz family protein in a gram-negative bacterium to produce the sugar.
C9. The composition of clause C8, wherein the wzz family protein is selected from the group consisting of: wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzz1, and wzz 2.
C10. The composition of clause C8, wherein the wzz family protein is wzzB.
C11. The composition of clause C8, wherein the wzz family protein is fepE.
C12. The composition of clause C8, wherein the wzz family proteins are wzzB and fepE.
C13. The composition of clause C8, wherein the wzz family protein is derived from salmonella enterica.
C14. The composition of clause C8, wherein the wzz family protein comprises a sequence selected from any one of the following: 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 SEQ ID NO.
C15. The composition of clause C8, wherein the wzz family protein comprises a sequence having at least 90% sequence identity to any one of the following sequences: 30, 31, 32, 33, 34.
C16. The composition of clause C8, wherein the wzz family protein comprises a sequence selected from any one of the following: 35, 36, 37, 38 and 39 SEQ ID NO.
C17. The composition according to clause C1, wherein the sugar is synthetically synthesized.
C18. The composition of any one of clauses C1-C17, wherein the sugar further comprises an e.coli R1 moiety.
C19. The composition of any one of clauses C1-C17, wherein the sugar further comprises an e.coli R2 moiety.
C20. The composition of any one of clauses C1-C17, wherein the sugar further comprises an e.coli R3 moiety.
C21. The composition of any one of clauses C1-C17, wherein the sugar further comprises an e.coli R4 moiety.
C22. The composition of any one of clauses C1-C17, wherein the saccharide further comprises an e.coli K-12 moiety.
C23. The composition of any one of clauses C1-C22, wherein the sugar further comprises a 3-deoxy-d-manno-oct-2-ketonic acid (KDO) moiety.
C24. The composition of any one of clauses C1 to C17, wherein the saccharide does not further comprise an e.coli R1 moiety.
C25. The composition of any one of clauses C1 to C17, wherein the saccharide does not further comprise an e.coli R2 moiety.
C26. The composition of any one of clauses C1 to C17, wherein the saccharide does not further comprise an e.coli R3 moiety.
C27. The composition of any one of clauses C1 to C17, wherein the saccharide does not further comprise an e.coli R4 moiety.
C28. The composition of any one of clauses C1 to C17, wherein the saccharide does not further comprise an e.coli K-12 moiety.
C29. The composition according to any one of clauses C1 to C22, wherein the saccharide does not further comprise a 3-deoxy-d-manno-oct-2-ketonic acid (KDO) moiety.
C30. The composition of any one of clauses C1 to C23, wherein the saccharide does not comprise lipid a.
C31. The composition of any one of clauses C1-C30, wherein the polysaccharide has a molecular weight of between 10kDa and 2,000kDa, or between 50kDa and 2,000 kDa.
C32. The composition of any one of clauses C1 to C31, wherein the average molecular weight of the saccharide is 20-40 kDa.
C33. The composition according to any one of clauses C1 to C32, wherein the average molecular weight of the saccharide is 40,000 to 60,000 kDa.
C34. The composition of any of clauses C1 to C33, wherein n is an integer from 31 to 90.
C35. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide is derived from e.
C36. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a saccharide according to any one of clauses C1 to clause C34 covalently bound to a carrier protein.
C37. A composition comprising a polypeptide derived from FimH, or a fragment thereof;and a conjugate according to any one of clauses C35 to clause C36, wherein the carrier protein is selected from any one of the following: poly (L-lysine), CRM197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid or exotoxin a from pseudomonas aeruginosa; detoxified exotoxin a (epa) of pseudomonas aeruginosa, Maltose Binding Protein (MBP), detoxified hemolysin a of staphylococcus aureus, aggregation factor a, aggregation factor B, cholera toxin B subunit (CTB), streptococcus pneumoniae pneumolysin and detoxified variants thereof, campylobacter jejuni AcrA, and campylobacter jejuni native glycoprotein.
C38. The composition according to any of clauses C35 to clause C37, wherein the carrier protein is CRM197。
C39. The composition according to any of clauses C35 to clause C37, wherein the carrier protein is Tetanus Toxoid (TT).
C40. The composition of any one of clauses C35 to clause C37, wherein the carrier protein is poly (L-lysine).
C41. The composition of any one of clauses C35 to clause C39, wherein the conjugate is prepared by reductive amination.
C42. The composition of any one of clauses C35 to clause C39, wherein the conjugate is prepared by CDAP chemistry.
C43. The composition of any one of clauses C35 to clause C39, wherein the conjugate is a single-ended conjugated saccharide.
C44. The composition of any one of clauses C35 to clause C39, wherein the saccharide is conjugated to a carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer.
C45. The composition of clause C44, wherein the saccharide is conjugated to the carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, wherein the saccharide is covalently attached to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently attached to the eTEC spacer through an amide linkage.
C46. The composition of any of clauses C44 to clause C45, wherein the CRM is197Comprising 2 to 20 or 4 to 16 lysine residues covalently linked to the polypeptide via an eTEC spacer.
C47. The composition of any one of clauses C35 to clause C46, wherein the ratio of saccharide: carrier protein (w/w) is between 0.2 and 4.
C48. The composition of any of clauses C35 to clause C46, wherein the ratio of sugar to protein is at least 0.5 and at most 2.
C49. The composition of any one of clauses C35 to clause C46, wherein the ratio of sugar to protein is between 0.4 and 1.7.
C50. The composition of any one of clauses C43 to clause C49, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ketonic acid (KDO) residue.
C51. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises a structure selected from the group consisting of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, wherein n is an integer from 1 to 10.
C52. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide according to any one of clauses C1 to clause C34, and a pharmaceutically acceptable diluent.
C53. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate according to any one of clauses C35 to clause C51, and a pharmaceutically acceptable diluent.
C54. The composition according to clause C53, comprising at most about 25% free sugar relative to the total amount of sugar in the composition.
C55. The composition of any one of clauses C52 to clause C53, further comprising an adjuvant.
C56. The composition of any of clauses C52 to clause C53, further comprising aluminum.
C57. The composition of any of clauses C52 to clause C53, further comprising QS-21.
C58. The composition of any one of clauses C52 to clause C53, further comprising a CpG oligonucleotide.
C59. The composition of any one of clauses C52 to clause C53, wherein the composition does not comprise an adjuvant.
C60. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide from e.coli conjugated to a carrier protein via a (2- ((2-oxyethyl) thio) ethyl) carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer via a carbamate linkage and the carrier protein is covalently linked to the eTEC spacer via an amide linkage.
C61. The composition of clause C60, wherein the saccharide is an O-antigen derived from escherichia coli.
C62. The composition of clause C60, further comprising a pharmaceutically acceptable excipient, carrier, or diluent.
C63. The composition of clause C60, wherein the saccharide is an O-antigen derived from escherichia coli.
C64. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide according to any one of clauses C1 to clause C17 conjugated to a carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer via a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer via an amide linkage.
C65. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and (i) a conjugate of e.coli O25B antigen covalently coupled to a carrier protein, (ii) an e.coli O1A antigen covalently coupled to a carrier protein, (iii) an e.coli O2 antigen covalently coupled to a carrier protein, and (iv) an O6 antigen covalently coupled to a carrier protein, wherein the e.coli O25B antigen comprises a structure of formula O25B, wherein n is an integer greater than 30.
C66. The composition of clause C65, wherein the carrier protein is selected from any one of the following: poly (L-lysine), CRM197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid or exotoxin a from pseudomonas aeruginosa; detoxified exotoxin a (epa) of pseudomonas aeruginosa, Maltose Binding Protein (MBP), detoxified hemolysin a of staphylococcus aureus, aggregation factor a, aggregation factor B, cholera toxin B subunit (CTB), streptococcus pneumoniae pneumolysin and detoxified variants thereof, campylobacter jejuni AcrA, and campylobacter jejuni native glycoprotein.
C67. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and (i) a conjugate of an e.coli O25B antigen covalently coupled to a carrier protein, (ii) an e.coli O4 antigen covalently coupled to a carrier protein, (iii) an e.coli O11 antigen covalently coupled to a carrier protein, and (iv) an O21 antigen covalently coupled to a carrier protein, wherein the e.coli O25B antigen comprises a structure of formula O75, wherein n is an integer greater than 30.
C68. The composition of clause C67, wherein the carrier protein is selected from any one of the following: poly (L-lysine), CRM 197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid or exotoxin a from pseudomonas aeruginosa; detoxified exotoxin a (epa), Maltose Binding Protein (MBP) of pseudomonas aeruginosa, detoxified hemolysin a, aggregation factor B, cholera toxin B subunit (CTB), streptococcus pneumoniae pneumolysin and detoxified variants thereof, campylobacter jejuni AcrA, and campylobacter jejuni native glycoprotein of staphylococcus aureus.
C69. A method of making a composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a saccharide conjugated to a carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, the method comprising the steps of: a) reacting a saccharide with 1,1 '-carbonyl-bis- (1,2, 4-triazole) (CDT) or 1, 1' -Carbonyldiimidazole (CDI) in an organic solvent to produce an activated saccharide; b) reacting the activated sugars with cystamine or cysteamine or a salt thereof to produce thiolated sugars; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more alpha-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α -haloacetamido groups of the activated carrier protein and/or (ii) a second capping reagent capable of capping unconjugated free thiol residues; thereby producing an eTEC-linked glycoconjugate, wherein the saccharide is derived from e.coli; also included is the expression of a polynucleotide encoding a polypeptide derived from FimH or a fragment thereof in a recombinant mammalian cell, and the isolation of the polypeptide or fragment thereof.
C70. The method of clause C69, including preparing the composition according to any one of clauses C1 to clause C34.
C71. The method of any one of clauses C69 to clause C70, wherein the capping step e) comprises reacting the thiolated saccharide-carrier protein conjugate with (i) N-acetyl-L-cysteine as a first capping reagent and/or (ii) iodoacetamide as a second capping reagent.
C72. The method of any of clauses C69 to clause C71, further comprising the step of complexing the saccharide by reaction with a triazole or imidazole to provide a complexed saccharide, wherein prior to step a), the complexed saccharide is shell frozen, lyophilized, and reconstituted in an organic solvent.
C73. The method according to any of clauses C69 to clause C72, further comprising purifying the thiolated polysaccharide produced in step C), wherein the purifying step comprises diafiltration.
C74. The method of any one of clauses C69 to clause C73, wherein the method further comprises purifying the eTEC-linked glycoconjugate by diafiltration.
C75. The method according to any of clauses C69 to clause C74, wherein the organic solvent in step a) is a polar aprotic solvent selected from any one of Dimethylsulfoxide (DMSO), Dimethylformamide (DMF), Dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU) and Hexamethylphosphoramide (HMPA), or a mixture thereof.
C76. A culture medium comprising KH2PO4、K2HPO4、(NH4)2SO4Sodium citrate, Na2SO4Aspartic acid, glucose, MgSO4、FeSO4-7H2O、Na2MoO4-2H2O、H3BO3、CoCl2-6H2O、CuCl2-2H2O、MnCl2-4H2O、ZnCl2And CaCl2-2H2O。
C77. The medium according to clause C76, wherein the medium is used for culturing e.
C78. The method of producing a saccharide according to any one of clauses C1 to clause C34, comprising culturing recombinant e.coli in a culture medium; producing the sugar by culturing the cell in the culture medium; whereby said cell produces said sugar.
C79. The method of clause C78, wherein the medium comprises a medium selected from KH2PO4、K2HPO4、(NH4)2SO4Sodium citrate, Na2SO4Aspartic acid, glucose, MgSO4、FeSO4-7H2O、Na2MoO4-2H2O、H3BO3、CoCl2-6H2O、CuCl2-2H2O、MnCl2-4H2O、ZnCl2And CaCl2-2H2O, or any of O.
C80. The method of clause C78, wherein the medium comprises soy hydrolysate.
C81. The method of clause C78, wherein the culture medium comprises yeast extract.
C82. The method of clause C78, wherein the medium does not further comprise soy hydrolysate and yeast extract.
C83. The method of clause C78, wherein the e.coli cell comprises a heterologous wzz family protein selected from any one of wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzz1, and wzz 2.
C84. The method of clause C78, wherein the e.coli cells comprise any one of the salmonella enterica wzz family proteins selected from the group consisting of wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzz1, and wzz 2.
C85. The method of clause C84, wherein the wzz family protein comprises a sequence selected from any one of the following: 20, 21, 22, 23, 24, 15, 16, 17, 18 and 19.
C86. The method of clause C78, wherein the culturing produces a yield of >120OD 600/mL.
C87. The method of clause C78, further comprising purifying the sugar.
C88. The method of clause C78, wherein the purifying step comprises any one of the following steps: dialysis, concentration procedures, diafiltration procedures, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultrafiltration, depth filtration and column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE and hydrophobic interaction chromatography).
C89. A method for inducing an immune response in a mammal comprising administering to the subject a composition according to any one of clauses C1 to clause C68.
C90. The method of clause C89, wherein the immune response comprises inducing serum antibodies against e.
C91. The method of clause C89, wherein the immune response comprises induction of anti-e.
C92. The method of clause C89, wherein the immune response comprises inducing bactericidal activity against e.
C93. The method of clause C89, wherein the immune response comprises induction of opsonophagocytic antibodies against e.
C94. The method of clause C89, wherein the immune response comprises a Geometric Mean Titer (GMT) level of at least 1,000 to 200,000 after initial administration.
C95. The method of clause C89, wherein the composition comprises a saccharide comprising the formula O25, wherein n is an integer from 40 to 100, wherein the immune response comprises a Geometric Mean Titer (GMT) level of at least 1,000 to 200,000 after initial administration.
C96. The method of clause C89, wherein the mammal is at risk of any one of the conditions selected from the group consisting of: urinary tract infections, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urinary sepsis, intra-abdominal infections, meningitis, complicated pneumonia, wound infections, infections associated after prostate biopsy, neonatal/infant sepsis, neutropenic fever and other bloodstream infections; pneumonia, bacteremia and sepsis.
C97. The method according to item C89, wherein the mammal has any one of the conditions selected from the group consisting of: urinary tract infections, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urinary sepsis, intra-abdominal infections, meningitis, complicated pneumonia, wound infections, infections associated after prostate biopsy, neonatal/infant sepsis, neutropenic fever and other bloodstream infections; pneumonia, bacteremia and sepsis.
C98. A method for (i) inducing an immune response against enteropathogenic escherichia coli in a subject, (ii) inducing an immune response against enteropathogenic escherichia coli in a subject, or (iii) inducing the production of opsonophagocytic antibodies specific for enteropathogenic escherichia coli in a subject, wherein the method comprises administering to the subject an effective amount of a composition according to any one of clauses C1 to clause C68.
C99. The method of clause C98, wherein the subject is at risk of developing a urinary tract infection.
C100. The method of clause C98, wherein the subject is at risk of developing bacteremia.
C101. The method of clause C98, wherein the subject is at risk of developing sepsis.
C102. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and (i) a conjugate of an e.coli O25B antigen covalently coupled to a carrier protein, (ii) an e.coli O1A antigen covalently coupled to a carrier protein, (iii) an e.coli O2 antigen covalently coupled to a carrier protein, and (iv) an O6 antigen covalently coupled to a carrier protein, wherein the e.coli O25B antigen comprises a structure of formula O25B, wherein n is an integer greater than 30.
C103. The composition of clause C102, wherein the carrier protein is selected from the group consisting of: poly (L-lysine), detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197Maltose Binding Protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, aggregation factor A, aggregation factor B, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Streptococcus pneumoniae pneumolysin and detoxified variants thereof, Campylobacter jejuni AcrA, and Campylobacter jejuni native glycoprotein.
C104. A method for (i) inducing an immune response against extraintestinal pathogenic escherichia coli in a subject, (ii) inducing an immune response against extraintestinal pathogenic escherichia coli in a subject, or (iii) inducing the production of opsonophagocytic antibodies specific for extraintestinal pathogenic escherichia coli in a subject, wherein the method comprises administering to the subject an effective amount of the composition of clause C1.
C105. The method of clause C104, wherein the subject is at risk of developing a urinary tract infection.
C106. The method of clause C104, wherein the subject is at risk of developing bacteremia.
C107. The method of clause C104, wherein the subject is at risk of developing sepsis.
C108. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide comprising an increase of at least 5 repeating units compared to the corresponding E.coli wild-type O-polysaccharide.
C109. The composition of clause C108, wherein the saccharide comprises the formula O25a, and the e.coli is e.coli serotype O25 a.
C110. The composition of clause C108, wherein the saccharide comprises the formula O25b, and the e.coli is e.coli serotype O25 b.
C111. The composition of clause C108, wherein the saccharide comprises formula O2 and the e.coli is e.coli serotype O2.
C112. The composition of clause C108, wherein the saccharide comprises formula O6 and the e.coli is e.coli serotype O6.
C113. The composition of clause C108, wherein the saccharide comprises formula O1 and the e.coli is e.coli serotype O1.
C114. The composition of clause C108, wherein the saccharide comprises formula O17 and the e.coli is e.coli serotype O17.
C115. The composition of clause C108, wherein the saccharide comprises a structure selected from the group consisting of: formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula 685, Formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O149, formula O156, formula O152, formula O175, formula O165, formula O175, formula O165, formula O162, formula O175, formula O168, formula O175, formula O165, formula O168, formula O175, formula O162, formula O168, formula O175, formula O168, formula O162, formula O175, formula O168, formula O175, formula, Formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 and formula O187, wherein n is an integer from 5 to 1000.
C116. The composition according to clause C108, wherein the e.coli is an e.coli serotype selected from the group consisting of: o1, O2, O3, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O6856856854, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, O4, o136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186 and O187.
C117. The composition of clause C108, wherein the sugar is produced by increasing the repeating units of O-polysaccharide in culture produced by a gram-negative bacterium, comprising overexpressing wzz family protein in a gram-negative bacterium to produce the sugar.
C118. The composition of clause C117, wherein the overexpressed wzz family protein is selected from the group consisting of: wzb, wzz, wzsf, wzst, fepE, wzfepe, wzz1, and wzz 2.
C119. The composition of clause C117, wherein the overexpressed wzz family protein is wzzB.
C120. The composition of clause C117, wherein the overexpressed wzz family protein is fepE.
C121. The composition of clause C117, wherein the overexpressed wzz family protein is wzzB and fepE.
C122. The composition according to clause C108, wherein the sugar is synthetically synthesized.
C123. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a saccharide according to clause C108 covalently bound to a carrier protein.
C124. The composition of clause C123, wherein the carrier protein is CRM197。
C125. The composition of clause C123, wherein the saccharide comprises a structure selected from the group consisting of: formula O, formula O25, formula O, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O149, formula O156, formula O152, formula O175, formula O165, formula O175, formula O165, formula O162, formula O175, formula O168, formula O175, formula O165, formula O168, formula O175, formula O162, formula O168, formula O175, formula O168, formula O162, formula O175, formula O168, formula O175, formula, Formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 and formula O187, wherein n is an integer from 5 to 1000.
C126. The composition of clause C123, wherein the saccharide comprises an increase of at least 5 repeating units compared to the corresponding wild-type O-polysaccharide.
C127. The composition according to clause C1, further comprising a pharmaceutically acceptable diluent.
C128. The composition according to clause C127, further comprising an adjuvant.
C129. The composition according to clause C127, further comprising aluminum.
C130. A composition according to clause C127, further comprising QS-21.
C131. The composition according to clause C127, wherein the composition does not comprise an adjuvant.
C132. A method for inducing an immune response in a subject, comprising administering to the subject a composition according to clause C127.
C133. The composition according to clause C123, further comprising a pharmaceutically acceptable diluent.
C134. A method for inducing an immune response in a subject comprising administering to the subject a composition according to clause C133.
C135. The method according to clause C132 or C134, wherein the immune response comprises inducing serum antibodies against e.
C136. The method of clause C135, wherein the anti-escherichia coli O-specific polysaccharide serum antibody is an IgG antibody.
C137. The method of clause C135, wherein the anti-e.coli O-specific polysaccharide serum antibodies are IgG antibodies having bactericidal activity against e.coli.
C138. An immunogenic composition comprising a polypeptide derived from FimH or a fragment thereof; and a saccharide derived from escherichia coli conjugated to a carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer via a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer via an amide linkage.
C139. The immunogenic composition of clause C138, further comprising a pharmaceutically acceptable excipient, carrier, or diluent.
C140. The immunogenic composition of clause C138, wherein the saccharide is an O-antigen derived from escherichia coli.
C141. The immunogenic composition of clause C138, wherein the saccharide comprises a structure selected from the group consisting of: formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula O14, formula 685, Formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O149, formula O156, formula O152, formula O175, formula O165, formula O175, formula O165, formula O162, formula O175, formula O168, formula O175, formula O165, formula O168, formula O175, formula O162, formula O168, formula O175, formula O168, formula O162, formula O175, formula O168, formula O175, formula, Formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 and formula O187, wherein n is an integer from 5 to 1000.
C142. The immunogenic composition according to clause C138, wherein the saccharide has a degree of O-acetylation of 75% to 100%.
C143. The immunogenic composition according to clause C138, wherein the carrier protein is CRM197。
C144. The immunogenic composition according to clause C143, wherein the CRM is197Comprising 2 to 20 lysine residues covalently linked to the polysaccharide through an eTEC spacer.
C145. The immunogenic composition according to clause C143, wherein the CRM is197Comprising 4 to 16 lysine residues covalently linked to the polysaccharide through an eTEC spacer.
C146. The immunogenic composition according to clause C138, further comprising an additional antigen.
C147. The immunogenic composition according to clause C138, further comprising an adjuvant.
C148. The immunogenic composition according to clause C147, wherein the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
C149. The immunogenic composition according to clause C138, wherein the composition does not comprise an adjuvant.
C150. An immunogenic composition comprising a polypeptide derived from FimH or a fragment thereof; and a glycoconjugate comprising an e.coli-derived saccharide conjugated to a carrier protein, wherein the glycoconjugate is prepared using reductive amination.
C151. The immunogenic composition of clause C150, further comprising a pharmaceutically acceptable excipient, carrier, or diluent.
C152. The immunogenic composition of clause C150, wherein the saccharide is an O-antigen derived from escherichia coli.
C153. The immunogenic composition of clause C150, wherein the saccharide comprises a structure selected from the group consisting of: formula O, formula O25, formula O, formula O, formula O, formula O, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O149, formula O156, formula O152, formula O175, formula O165, formula O175, formula O165, formula O162, formula O175, formula O168, formula O175, formula O165, formula O168, formula O175, formula O162, formula O168, formula O175, formula O168, formula O162, formula O175, formula O168, formula O175, formula, Formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186 and formula O187, wherein n is an integer from 5 to 1000.
C154. The immunogenic composition according to clause C150, wherein the saccharide has a degree of O-acetylation of 75% to 100%.
C155. The immunogenic composition according to clause C150, wherein the carrier protein is CRM197。
C156. The immunogenic composition according to clause C150, further comprising an additional antigen.
C157. The immunogenic composition according to clause C150, further comprising an adjuvant.
C158. The immunogenic composition according to clause C157, wherein the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
C159. The immunogenic composition according to clause C150, wherein the composition does not comprise an adjuvant.
C160. A method for inducing an immune response in a subject comprising administering to the subject a composition according to any one of clauses C138-C159.
C161. The method of clause C160, wherein the immune response comprises inducing serum antibodies against e.
C162. The method of clause C135, wherein the anti-escherichia coli O-specific polysaccharide serum antibody is an IgG antibody.
C163. The method of clause C135, wherein the anti-e.coli O-specific polysaccharide serum antibodies are IgG antibodies having bactericidal activity against e.coli.
C164. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a saccharide comprising a structure selected from any one of: formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4: K52, formula O4: K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6: K2; k13; k15, formula O6: K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A 18, formula O18B 18, formula O25 18, formula O18, formula O18, formula O18, formula O18, formula O18, formula O18, formula O18, formula 18, formula O18, formula 18, formula O18 formula 18, formula 18, formula O18 formula 18, formula O18, formula 18, formula O18, formula 18, formula O18, formula O18, formula 18 formula O18 formula 18, formula O18, formula 18 formula O18, formula 18O 18, formula 18O 18, formula O18, formula 18O 18, formula O18, formula 18O 18, formula 18O 18, formula 18O 18, formula 18O 18 formula 18O 18, formula 18O 18, formula 18O 18, formula 685, Formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O149, formula O152, formula O133, formula O123, formula O124, formula O168, formula O165, formula O143, formula O168, formula O143, formula O168, formula O143, formula O168, formula I, Formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, formula O187, wherein n is greater than the number of repeat units in the corresponding wild-type E.coli polypeptide.
C165. The composition of clause C164, wherein n is an integer from 31 to 100.
C166. The composition of clause C164, wherein the sugar comprises a structure according to any one of formula O1A, formula O1B, and formula O1C, formula O2, formula O6, and formula O25B.
C167. The composition of clause C164, wherein the saccharide is produced in a recombinant host cell that expresses a wzz family protein having at least 90% sequence identity to any one of the following sequences: 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 SEQ ID NO.
C168. The composition of clause C167, wherein the protein comprises any one of the following sequences: 30, 31, 32, 33, 34.
C169. The saccharide according to clause C164, wherein the saccharide is synthetically synthesized.
C170. A composition comprising a polypeptide derived from FimH, or a fragment thereof; and a conjugate comprising a carrier protein covalently bound to a saccharide comprising a structure selected from any one of: formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4: K52, formula O4: K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6: K2; k13; k15, formula O6: K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A 18, formula O18B 18, formula O18, formula O25 18, formula O18, formula O18, formula 18, formula O18, formula 18, formula 18, formula O18, formula 18, formula 18 formula O18, formula 18, formula 18 formula O18 formula 18, formula 18, formula 18 formula O18 formula 18, formula O18 formula, formula 18 formula O18 formula O18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula O18 formula, formula O18 formula O18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula O formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula 18 formula, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula 0111, formula O112, formula O113, formula O114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O149, formula O152, formula O133, formula O123, formula O124, formula O168, formula O165, formula O143, formula O168, formula O143, formula O168, formula O143, formula O168, formula I, Formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, formula O187, wherein n is an integer of 1 to 100.
C171. The composition of clause C170, wherein the sugar comprises any one of the following formulas O25b, formula O1A, formula O2, and formula O6.
C172. The composition of clause C170, wherein the saccharide further comprises any one of escherichia coli R1 portion, escherichia coli R2 portion, escherichia coli R3 portion, escherichia coli R4 portion, and escherichia coli K-12 portion.
C173. The composition of clause C170, wherein the saccharide does not further comprise any of escherichia coli R1 portion, escherichia coli R2 portion, escherichia coli R3 portion, escherichia coli R4 portion, and escherichia coli K-12 portion. The composition according to clause C170, wherein the saccharide does not further comprise the escherichia coli R2 portion.
C174. The composition according to clause C170, wherein the sugar further comprises a 3-deoxy-d-manno-oct-2-ketonic acid (KDO) moiety.
C175. The composition of clause C170, wherein the carrier protein is selected from any one of the following: CRM197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid or exotoxin a from pseudomonas aeruginosa; detoxified exotoxin a (epa) of pseudomonas aeruginosa, Maltose Binding Protein (MBP), detoxified hemolysin a of staphylococcus aureus, aggregation factor a, aggregation factor B, cholera toxin B subunit (CTB), streptococcus pneumoniae pneumolysin and detoxified variants thereof, campylobacter jejuni AcrA, and campylobacter jejuni native glycoprotein.
C176. The composition of clause C170, wherein the carrier protein is CRM197。
C177. The composition according to clause C170, wherein the carrier protein is tetanus toxoid.
C178. The composition according to clause C170, wherein the ratio of sugar to protein is at least 0.5 to at most 2.
C179. The composition according to clause C170, wherein the conjugate is prepared by reductive amination.
C180. The composition according to clause C170, wherein the saccharide is conjugated to the carrier protein via a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer.
C181. The composition of clause C170, wherein the saccharide is a single-end linked conjugated saccharide.
C182. The composition of clause C174, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ketonic acid (KDO) residue.
C183. The composition of clause C170, wherein the conjugate is prepared by CDAP chemistry.
C184. A composition comprising a polypeptide derived from FimH or a fragment thereof; and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising the formula O25b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising the formula O1A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising the formula O2, wherein n is an integer from 31 to 90, and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising the formula O6, wherein n is an integer from 31 to 90.
C185. The composition of clause C184, further comprising a conjugate comprising a carrier protein covalently bound to a saccharide comprising a structure selected from any one of: formula O15, formula O16, formula O17, formula O18, and formula O75, wherein n is an integer from 31 to 90.
C186. A composition according to clause C184, comprising up to 25% free sugar compared to the total amount of sugar in the composition.
C187. A method of eliciting an immune response against e.coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of clauses C184-C186.
C188. The method of clause C187, wherein the immune response comprises opsonophagocytic antibodies against e.
C189. The method of clause C187, wherein the immune response protects the mammal from e.
C190. A mammalian cell comprising (a) a first gene of interest encoding a polypeptide derived from escherichia coli, or a fragment thereof, wherein said gene is integrated between at least two Recombination Target Sites (RTS).
C191. The embodiment of clause C190, wherein the two RTS are chromosomally integrated within the NL1 locus or the NL2 locus.
C192. The embodiment of clause C190, wherein the first gene of interest further comprises a reporter gene, a gene encoding a protein that is difficult to express, an accessory gene, or a combination thereof.
C193. The embodiment of clause C190, further comprising a second gene of interest integrated within a second chromosomal locus different from the locus of (a), wherein the second gene of interest comprises a reporter gene, a gene encoding a protein that is difficult to express, an accessory gene, or a combination thereof.
SEQUENCE LISTING
<110> Pfizer Inc.
Donald, Robert G.K.
<120> E.coli compositions and methods thereof
<130> PC72517
<160> 109
<170> PatentIn version 3.5
<210> 1
<211> 300
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 1
Met Lys Arg Val Ile Thr Leu Phe Ala Val Leu Leu Met Gly Trp Ser
1 5 10 15
Val Asn Ala Trp Ser Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile
20 25 30
Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val
35 40 45
Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe
50 55 60
Cys His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln
65 70 75 80
Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val
85 90 95
Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro
100 105 110
Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu
115 120 125
Tyr Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly
130 135 140
Ser Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser
145 150 155 160
Asp Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val
165 170 175
Val Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr
180 185 190
Leu Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys
195 200 205
Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp
210 215 220
Ala Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln
225 230 235 240
Gly Val Gly Val Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn
245 250 255
Asn Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly
260 265 270
Leu Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn
275 280 285
Val Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln
290 295 300
<210> 2
<211> 279
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 2
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro
165 170 175
Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn
180 185 190
Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile
195 200 205
Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln
210 215 220
Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu
225 230 235 240
Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr
245 250 255
Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile
260 265 270
Gly Val Thr Phe Val Tyr Gln
275
<210> 3
<211> 156
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 3
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val
145 150 155
<210> 4
<211> 120
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 4
Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr
1 5 10 15
Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln
20 25 30
Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser
35 40 45
Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val
50 55 60
Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser
65 70 75 80
Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn
85 90 95
Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile
100 105 110
Ile Gly Val Thr Phe Val Tyr Gln
115 120
<210> 5
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 5
Val Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro
20 25 30
Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val
35 40 45
Asn Val Gly Gln Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys
50 55 60
His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg
65 70 75 80
Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys
85 90 95
Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg
100 105 110
Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr
115 120 125
Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser
130 135 140
Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp
145 150 155 160
Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val
165 170 175
Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu
180 185 190
Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala
195 200 205
Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala
210 215 220
Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly
225 230 235 240
Val Gly Val Gln Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn
245 250 255
Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu
260 265 270
Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val
275 280 285
Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly
290 295 300
Gly Gly Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys
305 310 315 320
Gly Gly His His His His His His His His
325 330
<210> 6
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 6
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro
20 25 30
Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val
35 40 45
Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys
50 55 60
His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg
65 70 75 80
Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys
85 90 95
Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg
100 105 110
Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr
115 120 125
Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser
130 135 140
Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp
145 150 155 160
Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val
165 170 175
Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu
180 185 190
Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala
195 200 205
Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala
210 215 220
Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly
225 230 235 240
Val Gly Val Gln Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn
245 250 255
Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu
260 265 270
Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val
275 280 285
Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly
290 295 300
Gly Gly Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys
305 310 315 320
Gly Gly His His His His His His His His
325 330
<210> 7
<211> 188
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 7
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro
20 25 30
Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val
35 40 45
Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys
50 55 60
His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg
65 70 75 80
Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys
85 90 95
Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg
100 105 110
Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr
115 120 125
Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser
130 135 140
Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp
145 150 155 160
Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val
165 170 175
Pro Thr Gly Gly His His His His His His His His
180 185
<210> 8
<211> 188
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 8
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro
20 25 30
Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val
35 40 45
Asn Val Gly Gln Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys
50 55 60
His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg
65 70 75 80
Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys
85 90 95
Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg
100 105 110
Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr
115 120 125
Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser
130 135 140
Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp
145 150 155 160
Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val
165 170 175
Pro Thr Gly Gly His His His His His His His His
180 185
<210> 9
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 9
Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys
1 5 10
<210> 10
<211> 241
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 10
Met Ser Asn Lys Asn Val Asn Val Arg Lys Ser Gln Glu Ile Thr Phe
1 5 10 15
Cys Leu Leu Ala Gly Ile Leu Met Phe Met Ala Met Met Val Ala Gly
20 25 30
Arg Ala Glu Ala Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr Pro
35 40 45
Ala Gly Gln Lys Gln Val Gln Leu Ala Val Thr Asn Asn Asp Glu Asn
50 55 60
Ser Thr Tyr Leu Ile Gln Ser Trp Val Glu Asn Ala Asp Gly Val Lys
65 70 75 80
Asp Gly Arg Phe Ile Val Thr Pro Pro Leu Phe Ala Met Lys Gly Lys
85 90 95
Lys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr Asn Asn Gln Leu Pro
100 105 110
Gln Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys Ala Ile Pro Ser
115 120 125
Met Asp Lys Ser Lys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile Ile
130 135 140
Ser Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys Leu Ala Leu Pro Pro
145 150 155 160
Asp Gln Ala Ala Glu Lys Leu Arg Phe Arg Arg Ser Ala Asn Ser Leu
165 170 175
Thr Leu Ile Asn Pro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu Asn
180 185 190
Ala Gly Thr Arg Val Leu Glu Asn Ala Leu Val Pro Pro Met Gly Glu
195 200 205
Ser Thr Val Lys Leu Pro Ser Asp Ala Gly Ser Asn Ile Thr Tyr Arg
210 215 220
Thr Ile Asn Asp Tyr Gly Ala Leu Thr Pro Lys Met Thr Gly Val Met
225 230 235 240
Glu
<210> 11
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 11
Asp Asn Lys Gln
1
<210> 12
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 12
Gly Gly Ser Gly Gly
1 5
<210> 13
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 13
Gly Gly Ser Ser Gly Gly
1 5
<210> 14
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 14
Gly Gly Ser Ser Gly Gly Gly
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 15
Gly Gly Gly Ser Ser Gly Gly Gly
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthesis of
<400> 16
Gly Gly Gly Ser Gly Ser Gly Gly Gly
1 5
<210> 17
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 17
Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly
1 5 10
<210> 18
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 18
Met Lys Arg Val Ile Thr Leu Phe Ala Val Leu Leu Met Gly Trp Ser
1 5 10 15
Val Asn Ala Trp Ser
20
<210> 19
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 19
Val Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 20
<211> 279
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 20
Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Asn Val Gly Gln
20 25 30
Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro
165 170 175
Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn
180 185 190
Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile
195 200 205
Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln
210 215 220
Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu
225 230 235 240
Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr
245 250 255
Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile
260 265 270
Gly Val Thr Phe Val Tyr Gln
275
<210> 21
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 21
Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly
1 5 10 15
His His His His His His His His
20
<210> 22
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 22
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 23
<211> 279
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 23
Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro
165 170 175
Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn
180 185 190
Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile
195 200 205
Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln
210 215 220
Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu
225 230 235 240
Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr
245 250 255
Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile
260 265 270
Gly Val Thr Phe Val Tyr Gln
275
<210> 24
<211> 160
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 24
Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
<210> 25
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 25
His His His His His His His His
1 5
<210> 26
<211> 160
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 26
Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Asn Val Gly Gln
20 25 30
Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
<210> 27
<211> 168
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 27
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
His His His His His His His His
165
<210> 28
<211> 279
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 28
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr
50 55 60
Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro
165 170 175
Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn
180 185 190
Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile
195 200 205
Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln
210 215 220
Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu
225 230 235 240
Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr
245 250 255
Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile
260 265 270
Gly Val Thr Phe Val Tyr Gln
275
<210> 29
<211> 279
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 29
Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly
1 5 10 15
Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln
20 25 30
Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr
35 40 45
Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ala Ala Tyr
50 55 60
Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Asn Gly Ser
65 70 75 80
Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn
85 90 95
Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val
100 105 110
Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val
115 120 125
Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe
130 135 140
Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly
145 150 155 160
Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro
165 170 175
Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn
180 185 190
Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile
195 200 205
Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln
210 215 220
Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu
225 230 235 240
Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr
245 250 255
Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile
260 265 270
Gly Val Thr Phe Val Tyr Gln
275
<210> 30
<211> 325
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 30
Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu
1 5 10 15
Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met
20 25 30
Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr
35 40 45
Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln
50 55 60
Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile
65 70 75 80
Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile
85 90 95
Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn
100 105 110
Gln Glu Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln
115 120 125
Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala
130 135 140
Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn
145 150 155 160
Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys
165 170 175
Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln
180 185 190
Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn
195 200 205
Gln Glu Gln Val Thr Lys Pro Gln Val Gln Gln Thr Glu Asp Val Thr
210 215 220
Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met Ile
225 230 235 240
Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Ser Asn Tyr Tyr Gln
245 250 255
Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp Leu
260 265 270
Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile Arg
275 280 285
Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu Leu
290 295 300
Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg
305 310 315 320
Asn Tyr Asn Ala Lys
325
<210> 31
<211> 326
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 31
Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn Asn Asp Pro Glu
1 5 10 15
Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met
20 25 30
Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr
35 40 45
Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln
50 55 60
Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile
65 70 75 80
Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile
85 90 95
Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn
100 105 110
Gln Asp Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln
115 120 125
Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala
130 135 140
Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn
145 150 155 160
Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys
165 170 175
Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln
180 185 190
Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn
195 200 205
Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile
210 215 220
Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met
225 230 235 240
Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr
245 250 255
Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp
260 265 270
Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile
275 280 285
Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu
290 295 300
Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu
305 310 315 320
Arg Asn Tyr Asn Ala Lys
325
<210> 32
<211> 326
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 32
Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu
1 5 10 15
Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met
20 25 30
Thr Ile Ile Ile Ser Val Val Val Ala Ile Ala Leu Ala Ile Gly Tyr
35 40 45
Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln
50 55 60
Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile
65 70 75 80
Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile
85 90 95
Gly Arg Phe Ser Phe Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn
100 105 110
Gln Lys Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln
115 120 125
Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Asp Ala
130 135 140
Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn
145 150 155 160
Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Leu Ala Leu Gly Arg Lys
165 170 175
Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln
180 185 190
Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn
195 200 205
Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile
210 215 220
Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met
225 230 235 240
Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr
245 250 255
Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Asn Leu Lys Val Asp Asp
260 265 270
Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile
275 280 285
Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu
290 295 300
Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu
305 310 315 320
Arg Asn Tyr Asn Ser Lys
325
<210> 33
<211> 326
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 33
Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu
1 5 10 15
Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met
20 25 30
Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr
35 40 45
Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln
50 55 60
Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile
65 70 75 80
Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile
85 90 95
Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn
100 105 110
Gln Glu Glu Arg Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln
115 120 125
Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala
130 135 140
Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn
145 150 155 160
Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys
165 170 175
Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln
180 185 190
Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn
195 200 205
Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile
210 215 220
Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met
225 230 235 240
Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr
245 250 255
Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp
260 265 270
Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Met Leu Pro Ile
275 280 285
Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu
290 295 300
Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu
305 310 315 320
Arg Asn Tyr Asn Ala Lys
325
<210> 34
<211> 327
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 34
Met Thr Val Asp Ser Asn Thr Ser Ser Gly Arg Gly Asn Asp Pro Glu
1 5 10 15
Gln Ile Asp Leu Ile Glu Leu Leu Leu Gln Leu Trp Arg Gly Lys Met
20 25 30
Thr Ile Ile Val Ala Val Ile Ile Ala Ile Leu Leu Ala Val Gly Tyr
35 40 45
Leu Met Ile Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln
50 55 60
Pro Asp Ala Ala Gln Val Ala Thr Tyr Thr Asn Ala Leu Asn Val Leu
65 70 75 80
Tyr Gly Gly Asn Ala Pro Lys Ile Ser Glu Val Gln Ala Asn Phe Ile
85 90 95
Ser Arg Phe Ser Ser Ala Phe Ser Ala Leu Ser Glu Val Leu Asp Asn
100 105 110
Gln Lys Glu Arg Glu Lys Leu Thr Ile Glu Gln Ser Val Lys Gly Gln
115 120 125
Ala Leu Pro Leu Ser Val Ser Tyr Val Ser Thr Thr Ala Glu Gly Ala
130 135 140
Gln Arg Arg Leu Ala Glu Tyr Ile Gln Gln Val Asp Glu Glu Val Ala
145 150 155 160
Lys Glu Leu Glu Val Asp Leu Lys Asp Asn Ile Thr Leu Gln Thr Lys
165 170 175
Thr Leu Gln Glu Ser Leu Glu Thr Gln Glu Val Val Ala Gln Glu Gln
180 185 190
Lys Asp Leu Arg Ile Lys Gln Ile Glu Glu Ala Leu Arg Tyr Ala Asp
195 200 205
Glu Ala Lys Ile Thr Gln Pro Gln Ile Gln Gln Thr Gln Asp Val Thr
210 215 220
Gln Asp Thr Met Phe Leu Leu Gly Ser Asp Ala Leu Lys Ser Met Ile
225 230 235 240
Gln Asn Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Ala Tyr Tyr Gln
245 250 255
Thr Lys Gln Thr Leu Leu Asp Ile Lys Asn Leu Lys Val Thr Ala Asp
260 265 270
Thr Val His Val Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Val Arg
275 280 285
Arg Asp Ser Pro Lys Thr Ala Ile Thr Leu Val Leu Ala Val Leu Leu
290 295 300
Gly Gly Met Ile Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg
305 310 315 320
Ser Tyr Lys Pro Lys Ala Leu
325
<210> 35
<211> 377
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 35
Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp
1 5 10 15
Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu
20 25 30
Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe
35 40 45
Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys
50 55 60
Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln
65 70 75 80
Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile
85 90 95
Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln
100 105 110
Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met
115 120 125
Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala
130 135 140
Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser
145 150 155 160
Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe
165 170 175
Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile
180 185 190
Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg
195 200 205
Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln
210 215 220
Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu
225 230 235 240
Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val
245 250 255
Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser
260 265 270
Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val
275 280 285
Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu
290 295 300
Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro
305 310 315 320
Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro
325 330 335
Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val
340 345 350
Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln
355 360 365
Asp Ala Met Met Ala Asp His Leu Val
370 375
<210> 36
<211> 377
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 36
Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Glu Ala His Phe Pro Glu
1 5 10 15
Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu
20 25 30
Ile Glu Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe
35 40 45
Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys
50 55 60
Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln
65 70 75 80
Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile
85 90 95
Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln
100 105 110
Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met
115 120 125
Asp Gln Leu Lys Glu Ala Lys Ile Asp Pro Leu Asp Leu His Arg Ala
130 135 140
Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser
145 150 155 160
Lys Lys Lys Asp Glu Ser Ala Leu Tyr Thr Ser Trp Thr Leu Ser Phe
165 170 175
Thr Ala Pro Thr Ser Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile
180 185 190
Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg
195 200 205
Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln
210 215 220
Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu
225 230 235 240
Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val
245 250 255
Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser
260 265 270
Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val
275 280 285
Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu
290 295 300
Val Glu Gln Leu Thr Lys Thr Asn Ile Asn Asp Val Asn Phe Thr Pro
305 310 315 320
Phe Lys Tyr Gln Leu Arg Pro Ser Leu Pro Val Lys Lys Asp Gly Gln
325 330 335
Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Val Gly Gly Met Val
340 345 350
Ala Cys Gly Gly Val Leu Leu Arg His Ala Met Ala Ser Arg Lys Gln
355 360 365
Asp Ala Met Met Ala Asp His Leu Val
370 375
<210> 37
<211> 377
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 37
Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp
1 5 10 15
Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu
20 25 30
Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe
35 40 45
Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys
50 55 60
Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln
65 70 75 80
Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile
85 90 95
Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln
100 105 110
Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met
115 120 125
Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala
130 135 140
Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser
145 150 155 160
Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe
165 170 175
Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile
180 185 190
Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg
195 200 205
Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln
210 215 220
Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu
225 230 235 240
Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val
245 250 255
Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser
260 265 270
Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val
275 280 285
Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu
290 295 300
Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro
305 310 315 320
Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro
325 330 335
Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val
340 345 350
Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln
355 360 365
Asp Ala Met Met Ala Asp His Leu Val
370 375
<210> 38
<211> 377
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 38
Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp
1 5 10 15
Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu
20 25 30
Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe
35 40 45
Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys
50 55 60
Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln
65 70 75 80
Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile
85 90 95
Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln
100 105 110
Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met
115 120 125
Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala
130 135 140
Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser
145 150 155 160
Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe
165 170 175
Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile
180 185 190
Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg
195 200 205
Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln
210 215 220
Asp Arg Ile Lys Met Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu
225 230 235 240
Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val
245 250 255
Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser
260 265 270
Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val
275 280 285
Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu
290 295 300
Val Glu Gln Leu Thr Lys Ala Asn Ile Asn Asp Val Asn Phe Thr Pro
305 310 315 320
Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro
325 330 335
Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val
340 345 350
Ala Cys Gly Ser Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln
355 360 365
Asp Ala Met Met Ala Asp His Leu Val
370 375
<210> 39
<211> 378
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 39
Met Pro Ser Leu Asn Val Lys Gln Glu Lys Asn Gln Ser Phe Ala Gly
1 5 10 15
Tyr Ser Leu Pro Pro Ala Asn Ser His Glu Ile Asp Leu Phe Ser Leu
20 25 30
Ile Glu Val Leu Trp Gln Ala Lys Arg Arg Ile Leu Ala Thr Val Phe
35 40 45
Ala Phe Ala Cys Val Gly Leu Leu Leu Ser Phe Leu Leu Pro Gln Lys
50 55 60
Trp Thr Ser Gln Ala Ile Val Thr Pro Ala Glu Ser Val Gln Trp Gln
65 70 75 80
Gly Leu Glu Arg Thr Leu Thr Ala Leu Arg Val Leu Asp Met Glu Val
85 90 95
Ser Val Asp Arg Gly Ser Val Phe Asn Leu Phe Ile Lys Lys Phe Ser
100 105 110
Ser Pro Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met
115 120 125
Asp Gln Leu Lys Gly Ala Gln Ile Asp Glu Gln Asp Leu His Arg Ala
130 135 140
Ile Val Leu Leu Ser Glu Lys Met Lys Ala Val Asp Ser Asn Val Gly
145 150 155 160
Lys Lys Asn Glu Thr Ser Leu Phe Thr Ser Trp Thr Leu Ser Phe Thr
165 170 175
Ala Pro Thr Arg Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile Gln
180 185 190
Tyr Ile Ser Asp Ile Val Val Lys Glu Thr Leu Glu Asn Ile Arg Asn
195 200 205
Gln Leu Glu Ile Lys Thr Arg Tyr Glu Gln Glu Lys Leu Ala Met Asp
210 215 220
Arg Val Arg Leu Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu His
225 230 235 240
Tyr Ser Leu Glu Ile Ala Asn Ala Ala Gly Ile Lys Arg Pro Val Tyr
245 250 255
Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser Leu
260 265 270
Gly Ala Asp Gly Ile Ser Arg Lys Leu Glu Ile Glu Lys Gly Val Thr
275 280 285
Asp Val Ala Glu Ile Asp Gly Asp Leu Arg Asn Arg Gln Tyr His Val
290 295 300
Glu Gln Leu Ala Ala Met Asn Val Ser Asp Val Lys Phe Thr Pro Phe
305 310 315 320
Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro Gly
325 330 335
Lys Ala Ile Ile Ile Ile Leu Ala Ala Leu Ile Gly Gly Met Met Ala
340 345 350
Cys Gly Gly Val Leu Leu Arg His Ala Met Val Ser Arg Lys Met Glu
355 360 365
Asn Ala Leu Ala Ile Asp Glu Arg Leu Val
370 375
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 40
gaagcaaacc gtacgcgtaa ag 22
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 41
cgaccagctc ttacacggcg 20
<210> 42
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 42
gaaataggac cactaataaa tacacaaatt aataac 36
<210> 43
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 43
ataattgacg atccggttgc c 21
<210> 44
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> Synthesis of
<400> 44
gctatttacg ccctgattgt cttttgt 27
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Synthesis of
<400> 45
attgagaacc tgcgtaaacg gc 22
<210> 46
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Synthesis of
<400> 46
tgaagagcgg ttcagataac ttcc 24
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 47
cgatccggaa acctcctaca c 21
<210> 48
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 48
gattattcgc gcaacgctaa acagat 26
<210> 49
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 49
tgatcattga cgatccggta gcc 23
<210> 50
<211> 70
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 50
cggtagctgt aaagccaggg gcggtagcgt ggtttaaacc caagcaacag atcggcgtcg 60
tcggtatgga 70
<210> 51
<211> 78
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 51
agcttccata ccgacgacgc cgatctgttg cttgggttta aaccacgcta ccgcccctgg 60
ctttacagct accgagct 78
<210> 52
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 52
ggtagctgta aagccagggg cggtagcgtg 30
<210> 53
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 53
ccataccgac gacgccgatc tgttgcttgg 30
<210> 54
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 54
Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly
1 5 10 15
Ser Thr Gly
<210> 55
<211> 23
<212> PRT
<213> Intelligent people
<400> 55
Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe
1 5 10 15
Leu Leu Pro Gly Ser Leu Gly
20
<210> 56
<211> 18
<212> PRT
<213> Intelligent people
<400> 56
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val
1 5 10 15
Arg Ala
<210> 57
<211> 25
<212> PRT
<213> human respiratory syncytial virus type A (A2 strain)
<400> 57
Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Gly
20 25
<210> 58
<211> 15
<212> PRT
<213> influenza A virus (A strain/Japan/305/1957H 2N 2)
<400> 58
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly
1 5 10 15
<210> 59
<211> 207
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 59
Met Glu Gly Met Asp Pro Leu Ala Val Leu Ala Glu Ser Arg Leu Leu
1 5 10 15
Pro Leu Leu Thr Val Arg Gly Gly Glu Asp Leu Ala Gly Leu Ala Thr
20 25 30
Val Leu Glu Leu Met Gly Val Gly Ala Leu Glu Ile Thr Leu Arg Thr
35 40 45
Glu Lys Gly Leu Glu Ala Leu Lys Ala Leu Arg Lys Ser Gly Leu Leu
50 55 60
Leu Gly Ala Gly Thr Val Arg Ser Pro Lys Glu Ala Glu Ala Ala Leu
65 70 75 80
Glu Ala Gly Ala Ala Phe Leu Val Ser Pro Gly Leu Leu Glu Glu Val
85 90 95
Ala Ala Leu Ala Gln Ala Arg Gly Val Pro Tyr Leu Pro Gly Val Leu
100 105 110
Thr Pro Thr Glu Val Glu Arg Ala Leu Ala Leu Gly Leu Ser Ala Leu
115 120 125
Lys Phe Phe Pro Ala Glu Pro Phe Gln Gly Val Arg Val Leu Arg Ala
130 135 140
Tyr Ala Glu Val Phe Pro Glu Val Arg Phe Leu Pro Thr Gly Gly Ile
145 150 155 160
Lys Glu Glu His Leu Pro His Tyr Ala Ala Leu Pro Asn Leu Leu Ala
165 170 175
Val Gly Gly Ser Trp Leu Leu Gln Gly Asp Leu Ala Ala Val Met Lys
180 185 190
Lys Val Lys Ala Ala Lys Ala Leu Leu Ser Pro Gln Ala Pro Gly
195 200 205
<210> 60
<211> 156
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 60
Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp
1 5 10 15
Met Ala Glu Ala Ala Ile Arg Thr Leu Lys Ala Leu Ser Pro Asn Ile
20 25 30
Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala
35 40 45
Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu
50 55 60
Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala
65 70 75 80
Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile
85 90 95
Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Asp Glu Leu Asp
100 105 110
Ile Leu Ala Leu Val Arg Ala Ile Glu His Ala Ala Asn Val Tyr Tyr
115 120 125
Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu
130 135 140
Arg Gln Gly Arg Glu Asp Ala Gly Pro Ala Arg Glu
145 150 155
<210> 61
<211> 156
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 61
Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp
1 5 10 15
Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile
20 25 30
Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala
35 40 45
Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu
50 55 60
Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala
65 70 75 80
Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile
85 90 95
Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys
100 105 110
Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr
115 120 125
Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu
130 135 140
Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu
145 150 155
<210> 62
<211> 209
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 62
Met Ser Thr Ile Asn Asn Gln Leu Lys Ala Leu Lys Val Ile Pro Val
1 5 10 15
Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu
20 25 30
Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala
35 40 45
Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu
50 55 60
Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys
65 70 75 80
Glu Ala Gly Ala Thr Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr
85 90 95
Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn
100 105 110
Asn Pro Ser Thr Val Glu Ala Ala Leu Glu Met Gly Leu Thr Thr Leu
115 120 125
Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser
130 135 140
Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile
145 150 155 160
Thr Pro Ser Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala
165 170 175
Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Thr Asn Gly Glu
180 185 190
Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn
195 200 205
Pro
<210> 63
<211> 114
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 63
Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Thr Asp Val Pro
1 5 10 15
Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser
20 25 30
Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu
35 40 45
Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser
50 55 60
Ile Gly Gly Ile Glu Pro Ser Lys Asn Arg Asp His Ser Ala Val Leu
65 70 75 80
Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr
85 90 95
Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr
100 105 110
Thr Phe
<210> 64
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 64
Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala
20 25 30
Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg
115 120 125
Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala
130 135 140
Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 65
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 65
Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu
1 5 10 15
Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe
20 25 30
Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala
35 40 45
Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile
50 55 60
Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val
65 70 75 80
Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile
85 90 95
Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met
100 105 110
Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu
115 120 125
Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met
130 135 140
Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn
145 150 155 160
Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly
165 170 175
Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys
180 185 190
Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu
195 200 205
<210> 66
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 66
Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala
20 25 30
Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu
115 120 125
Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala
130 135 140
Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 67
<211> 177
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 67
Met Phe Thr Lys Ser Gly Asp Asp Gly Asn Thr Asn Val Ile Asn Lys
1 5 10 15
Arg Val Gly Lys Asp Ser Pro Leu Val Asn Phe Leu Gly Asp Leu Asp
20 25 30
Glu Leu Asn Ser Phe Ile Gly Phe Ala Ile Ser Lys Ile Pro Trp Glu
35 40 45
Asp Met Lys Lys Asp Leu Glu Arg Val Gln Val Glu Leu Phe Glu Ile
50 55 60
Gly Glu Asp Leu Ser Thr Gln Ser Ser Lys Lys Lys Ile Asp Glu Ser
65 70 75 80
Tyr Val Leu Trp Leu Leu Ala Ala Thr Ala Ile Tyr Arg Ile Glu Ser
85 90 95
Gly Pro Val Lys Leu Phe Val Ile Pro Gly Gly Ser Glu Glu Ala Ser
100 105 110
Val Leu His Val Thr Arg Ser Val Ala Arg Arg Val Glu Arg Asn Ala
115 120 125
Val Lys Tyr Thr Lys Glu Leu Pro Glu Ile Asn Arg Met Ile Ile Val
130 135 140
Tyr Leu Asn Arg Leu Ser Ser Leu Leu Phe Ala Met Ala Leu Val Ala
145 150 155 160
Asn Lys Arg Arg Asn Gln Ser Glu Lys Ile Tyr Glu Ile Gly Lys Ser
165 170 175
Trp
<210> 68
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 68
Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala
20 25 30
Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu
115 120 125
Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala
130 135 140
Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 69
<211> 201
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 69
Met Gly His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser Ala
1 5 10 15
Leu Arg Ile Gly Ile Val His Ala Arg Trp Asn Lys Thr Ile Ile Met
20 25 30
Pro Leu Leu Ile Gly Thr Ile Ala Lys Leu Leu Glu Cys Gly Val Lys
35 40 45
Ala Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu Leu Pro
50 55 60
Ile Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr Pro Ser
65 70 75 80
Ser Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser Ser Thr Thr
85 90 95
Asp Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr Gly Pro Phe
100 105 110
Asp Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly Glu Thr Met His
115 120 125
Phe Glu Tyr Ile Ala Asp Ser Val Ser His Gly Leu Met Arg Val Gln
130 135 140
Leu Asp Thr Gly Val Pro Val Ile Phe Gly Val Leu Thr Val Leu Thr
145 150 155 160
Asp Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Glu Gly Ser His Asn
165 170 175
His Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Met Gly Val Arg Arg
180 185 190
Arg Asp Trp Ala Ala Gly Lys Thr Glu
195 200
<210> 70
<211> 237
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 70
Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly
1 5 10 15
Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val
20 25 30
Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly
35 40 45
Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala
50 55 60
Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu
65 70 75 80
Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Gln Leu Gly
85 90 95
Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu
100 105 110
Lys Thr Val Ala Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His
115 120 125
Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu
130 135 140
Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly
145 150 155 160
Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr
165 170 175
Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg
180 185 190
His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Arg Glu Tyr
195 200 205
Glu Ala Ala Phe Met Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly
210 215 220
Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala
225 230 235
<210> 71
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 71
Met Gly Met Lys Glu Lys Phe Val Leu Ile Ile Thr His Gly Asp Phe
1 5 10 15
Gly Lys Gly Leu Leu Ser Gly Ala Glu Val Ile Ile Gly Lys Gln Glu
20 25 30
Asn Val His Thr Val Gly Leu Asn Leu Gly Asp Asn Ile Glu Lys Val
35 40 45
Ala Lys Glu Val Met Arg Ile Ile Ile Ala Lys Leu Ala Glu Asp Lys
50 55 60
Glu Ile Ile Ile Val Val Asp Leu Phe Gly Gly Ser Pro Phe Asn Ile
65 70 75 80
Ala Leu Glu Met Met Lys Thr Phe Asp Val Lys Val Ile Thr Gly Ile
85 90 95
Asn Met Pro Met Leu Val Glu Leu Leu Thr Ser Ile Asn Val Tyr Asp
100 105 110
Thr Thr Glu Leu Leu Glu Asn Ile Ser Lys Ile Gly Lys Asp Gly Ile
115 120 125
Lys Val Ile Glu Lys Ser Ser Leu Lys Met
130 135
<210> 72
<211> 154
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 72
Met Lys Tyr Asp Gly Ser Lys Leu Arg Ile Gly Ile Leu His Ala Arg
1 5 10 15
Trp Asn Leu Glu Ile Ile Ala Ala Leu Val Ala Gly Ala Ile Lys Arg
20 25 30
Leu Gln Glu Phe Gly Val Lys Ala Glu Asn Ile Ile Ile Glu Thr Val
35 40 45
Pro Gly Ser Phe Glu Leu Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys
50 55 60
Gln Lys Arg Leu Gly Lys Pro Leu Asp Ala Ile Ile Pro Ile Gly Val
65 70 75 80
Leu Ile Lys Gly Ser Thr Met His Phe Glu Tyr Ile Cys Asp Ser Thr
85 90 95
Thr His Gln Leu Met Lys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile
100 105 110
Phe Gly Val Leu Thr Cys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala
115 120 125
Gly Leu Ile Glu Gly Lys Met His Asn His Gly Glu Asp Trp Gly Ala
130 135 140
Ala Ala Val Glu Met Ala Thr Lys Phe Asn
145 150
<210> 73
<211> 164
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 73
Met Ala Val Lys Gly Leu Gly Glu Val Asp Gln Lys Tyr Asp Gly Ser
1 5 10 15
Lys Leu Arg Ile Gly Ile Leu His Ala Arg Trp Asn Arg Lys Ile Ile
20 25 30
Leu Ala Leu Val Ala Gly Ala Val Leu Arg Leu Leu Glu Phe Gly Val
35 40 45
Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Pro Gly Ser Phe Glu Leu
50 55 60
Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Gln Lys Arg Leu Gly Lys
65 70 75 80
Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Leu Ile Lys Gly Ser Thr
85 90 95
Met His Phe Glu Tyr Ile Cys Asp Ser Thr Thr His Gln Leu Met Lys
100 105 110
Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Phe Gly Val Leu Thr Cys
115 120 125
Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Gly Leu Ile Glu Gly Lys
130 135 140
Met His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala
145 150 155 160
Thr Lys Phe Asn
<210> 74
<211> 175
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 74
Met Gly Ala Asn Trp Tyr Leu Asp Asn Glu Ser Ser Arg Leu Ser Phe
1 5 10 15
Thr Ser Thr Lys Asn Ala Asp Ile Ala Glu Val His Arg Phe Leu Val
20 25 30
Leu His Gly Lys Val Asp Pro Lys Gly Leu Ala Glu Val Glu Val Glu
35 40 45
Thr Glu Ser Ile Ser Thr Gly Ile Pro Leu Arg Asp Met Leu Leu Arg
50 55 60
Val Leu Val Phe Gln Val Ser Lys Phe Pro Val Ala Gln Ile Asn Ala
65 70 75 80
Gln Leu Asp Met Arg Pro Ile Asn Asn Leu Ala Pro Gly Ala Gln Leu
85 90 95
Glu Leu Arg Leu Pro Leu Thr Val Ser Leu Arg Gly Lys Ser His Ser
100 105 110
Tyr Asn Ala Glu Leu Leu Ala Thr Arg Leu Asp Glu Arg Arg Phe Gln
115 120 125
Val Val Thr Leu Glu Pro Leu Val Ile His Ala Gln Asp Phe Asp Met
130 135 140
Val Arg Ala Phe Asn Ala Leu Arg Leu Val Ala Gly Leu Ser Ala Val
145 150 155 160
Ser Leu Ser Val Pro Val Gly Ala Val Leu Ile Phe Thr Ala Arg
165 170 175
<210> 75
<211> 208
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 75
Met Thr Asp Tyr Ile Arg Asp Gly Ser Ala Ile Lys Ala Leu Ser Phe
1 5 10 15
Ala Ile Ile Leu Ala Glu Ala Asp Leu Arg His Ile Pro Gln Asp Leu
20 25 30
Gln Arg Leu Ala Val Arg Val Ile His Ala Cys Gly Met Val Asp Val
35 40 45
Ala Asn Asp Leu Ala Phe Ser Glu Gly Ala Gly Lys Ala Gly Arg Asn
50 55 60
Ala Leu Leu Ala Gly Ala Pro Ile Leu Cys Asp Ala Arg Met Val Ala
65 70 75 80
Glu Gly Ile Thr Arg Ser Arg Leu Pro Ala Asp Asn Arg Val Ile Tyr
85 90 95
Thr Leu Ser Asp Pro Ser Val Pro Glu Leu Ala Lys Lys Ile Gly Asn
100 105 110
Thr Arg Ser Ala Ala Ala Leu Asp Leu Trp Leu Pro His Ile Glu Gly
115 120 125
Ser Ile Val Ala Ile Gly Asn Ala Pro Thr Ala Leu Phe Arg Leu Phe
130 135 140
Glu Leu Leu Asp Ala Gly Ala Pro Lys Pro Ala Leu Ile Ile Gly Met
145 150 155 160
Pro Val Gly Phe Val Gly Ala Ala Glu Ser Lys Asp Glu Leu Ala Ala
165 170 175
Asn Ser Arg Gly Val Pro Tyr Val Ile Val Arg Gly Arg Arg Gly Gly
180 185 190
Ser Ala Met Thr Ala Ala Ala Val Asn Ala Leu Ala Ser Glu Arg Glu
195 200 205
<210> 76
<211> 128
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 76
Met Ile Thr Val Phe Gly Leu Lys Ser Lys Leu Ala Pro Arg Arg Glu
1 5 10 15
Lys Leu Ala Glu Val Ile Tyr Ser Ser Leu His Leu Gly Leu Asp Ile
20 25 30
Pro Lys Gly Lys His Ala Ile Arg Phe Leu Cys Leu Glu Lys Glu Asp
35 40 45
Phe Tyr Tyr Pro Phe Asp Arg Ser Asp Asp Tyr Thr Val Ile Glu Ile
50 55 60
Asn Leu Met Ala Gly Arg Ser Glu Glu Thr Lys Met Leu Leu Ile Phe
65 70 75 80
Leu Leu Phe Ile Ala Leu Glu Arg Lys Leu Gly Ile Arg Ala His Asp
85 90 95
Val Glu Ile Thr Ile Lys Glu Gln Pro Ala His Cys Trp Gly Phe Arg
100 105 110
Gly Arg Thr Gly Asp Ser Ala Arg Asp Leu Asp Tyr Asp Ile Tyr Val
115 120 125
<210> 77
<211> 235
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 77
Met Gly Ser Asp Leu Gln Lys Leu Gln Arg Phe Ser Thr Cys Asp Ile
1 5 10 15
Ser Asp Gly Leu Leu Asn Val Tyr Asn Ile Pro Thr Gly Gly Tyr Phe
20 25 30
Pro Asn Leu Thr Ala Ile Ser Pro Pro Gln Asn Ser Ser Ile Val Gly
35 40 45
Thr Ala Tyr Thr Val Leu Phe Ala Pro Ile Asp Asp Pro Arg Pro Ala
50 55 60
Val Asn Tyr Ile Asp Ser Val Pro Pro Asn Ser Ile Leu Val Leu Ala
65 70 75 80
Leu Glu Pro His Leu Gln Ser Gln Phe His Pro Phe Ile Lys Ile Thr
85 90 95
Gln Ala Met Tyr Gly Gly Leu Met Ser Thr Arg Ala Gln Tyr Leu Lys
100 105 110
Ser Asn Gly Thr Val Val Phe Gly Arg Ile Arg Asp Val Asp Glu His
115 120 125
Arg Thr Leu Asn His Pro Val Phe Ala Tyr Gly Val Gly Ser Cys Ala
130 135 140
Pro Lys Ala Val Val Lys Ala Val Gly Thr Asn Val Gln Leu Lys Ile
145 150 155 160
Leu Thr Ser Asp Gly Val Thr Gln Thr Ile Cys Pro Gly Asp Tyr Ile
165 170 175
Ala Gly Asp Asn Asn Gly Ile Val Arg Ile Pro Val Gln Glu Thr Asp
180 185 190
Ile Ser Lys Leu Val Thr Tyr Ile Glu Lys Ser Ile Glu Val Asp Arg
195 200 205
Leu Val Ser Glu Ala Ile Lys Asn Gly Leu Pro Ala Lys Ala Ala Gln
210 215 220
Thr Ala Arg Arg Met Val Leu Lys Asp Tyr Ile
225 230 235
<210> 78
<211> 162
<212> PRT
<213> Artificial sequence
<220>
<223> Synthesis of
<400> 78
Met Ser Gly Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Tyr Glu
1 5 10 15
Leu Lys Gln Ala Ile Ile Ala Phe Leu Lys Met Thr Gly His Glu Pro
20 25 30
Ile Asp Cys Gly Ala Leu Arg Tyr Asp Ala Asp Asp Asp Tyr Pro Ala
35 40 45
Phe Cys Ile Ala Ala Ala Thr Arg Thr Val Ala Asp Pro Gly Ser Leu
50 55 60
Gly Ile Val Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn
65 70 75 80
Lys Val Pro Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Gln Thr Ala
85 90 95
Ala Leu Ala Arg Glu His Asn Asn Ala Gln Leu Ile Gly Ile Gly Gly
100 105 110
Arg Met His Thr Leu Glu Glu Ala Leu Arg Ile Val Lys Ala Phe Val
115 120 125
Thr Thr Pro Trp Ser Lys Ala Gln Arg His Gln Arg Arg Ile Asp Ile
130 135 140
Leu Ala Glu Tyr Glu Arg Thr His Glu Ala Pro Pro Val Pro Gly Ala
145 150 155 160
Pro Ala
<210> 79
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 79
Met Gly Asp Asp Ala Arg Ile Ala Ala Ile Gly Asp Val Asp Glu Leu
1 5 10 15
Asn Ser Gln Ile Gly Val Leu Leu Ala Glu Pro Leu Pro Asp Asp Val
20 25 30
Arg Ala Ala Leu Ser Ala Ile Gln His Asp Leu Phe Asp Leu Gly Gly
35 40 45
Glu Leu Cys Ile Pro Gly His Ala Ala Ile Thr Glu Asp His Leu Leu
50 55 60
Arg Leu Ala Leu Trp Leu Val His Tyr Asn Gly Gln Leu Pro Pro Leu
65 70 75 80
Glu Glu Phe Ile Leu Pro Gly Gly Ala Arg Gly Ala Ala Leu Ala His
85 90 95
Val Cys Arg Thr Val Cys Arg Arg Ala Glu Arg Ser Ile Lys Ala Leu
100 105 110
Gly Ala Ser Glu Pro Leu Asn Ile Ala Pro Ala Ala Tyr Val Asn Leu
115 120 125
Leu Ser Asp Leu Leu Phe Val Leu Ala Arg Val Leu Asn Arg Ala Ala
130 135 140
Gly Gly Ala Asp Val Leu Trp Asp Arg Thr Arg Ala His
145 150 155
<210> 80
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 80
Met Ile Leu Ser Ala Glu Gln Ser Phe Thr Leu Arg His Pro His Gly
1 5 10 15
Gln Ala Ala Ala Leu Ala Phe Val Arg Glu Pro Ala Ala Ala Leu Ala
20 25 30
Gly Val Gln Arg Leu Arg Gly Leu Asp Ser Asp Gly Glu Gln Val Trp
35 40 45
Gly Glu Leu Leu Val Arg Val Pro Leu Leu Gly Glu Val Asp Leu Pro
50 55 60
Phe Arg Ser Glu Ile Val Arg Thr Pro Gln Gly Ala Glu Leu Arg Pro
65 70 75 80
Leu Thr Leu Thr Gly Glu Arg Ala Trp Val Ala Val Ser Gly Gln Ala
85 90 95
Thr Ala Ala Glu Gly Gly Glu Met Ala Phe Ala Phe Gln Phe Gln Ala
100 105 110
His Leu Ala Thr Pro Glu Ala Glu Gly Glu Gly Gly Ala Ala Phe Glu
115 120 125
Val Met Val Gln Ala Ala Ala Gly Val Thr Leu Leu Leu Val Ala Met
130 135 140
Ala Leu Pro Gln Gly Leu Ala Ala Gly Leu Pro Pro Ala
145 150 155
<210> 81
<211> 156
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 81
Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp
1 5 10 15
Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile
20 25 30
Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala
35 40 45
Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu
50 55 60
Gly Met Pro Gly Lys Lys Glu Lys Asp Lys Val Cys Ala His Glu Ala
65 70 75 80
Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile
85 90 95
Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys
100 105 110
Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr
115 120 125
Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu
130 135 140
Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu
145 150 155
<210> 82
<211> 209
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 82
Met Asp Asp Ile Asn Asn Gln Leu Lys Arg Leu Lys Val Ile Pro Val
1 5 10 15
Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu
20 25 30
Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala
35 40 45
Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu
50 55 60
Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys
65 70 75 80
Glu Ala Gly Ala Asp Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr
85 90 95
Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn
100 105 110
Asn Pro Ser Thr Val Glu Gln Ala Leu Glu Met Gly Leu Thr Thr Leu
115 120 125
Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser
130 135 140
Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile
145 150 155 160
Thr Pro Asp Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala
165 170 175
Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Arg Asn Gly Glu
180 185 190
Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn
195 200 205
Pro
<210> 83
<211> 114
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 83
Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro
1 5 10 15
Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser
20 25 30
Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu
35 40 45
Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser
50 55 60
Ile Gly Gly Ile Glu Pro Asp Lys Asn Arg Asp His Ser Ala Val Leu
65 70 75 80
Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr
85 90 95
Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr
100 105 110
Thr Phe
<210> 84
<211> 114
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 84
Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro
1 5 10 15
Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser
20 25 30
Glu Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu
35 40 45
Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser
50 55 60
Ile Gly Gly Ile Glu Pro Asp Lys Asn Glu Asp His Ser Ala Val Leu
65 70 75 80
Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr
85 90 95
Ile His Phe Val Asp Leu Asp Gly Asp Asp Val Gly Trp Asn Gly Thr
100 105 110
Thr Phe
<210> 85
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 85
Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala
20 25 30
Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg
115 120 125
Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala
130 135 140
Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 86
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 86
Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala
20 25 30
Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu
115 120 125
Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala
130 135 140
Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 87
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 87
Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu
1 5 10 15
Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe
20 25 30
Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala
35 40 45
Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile
50 55 60
Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val
65 70 75 80
Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile
85 90 95
Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met
100 105 110
Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu
115 120 125
Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met
130 135 140
Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn
145 150 155 160
Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly
165 170 175
Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys
180 185 190
Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu
195 200 205
<210> 88
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 88
Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu
1 5 10 15
Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe
20 25 30
Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala
35 40 45
Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile
50 55 60
Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val
65 70 75 80
Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile
85 90 95
Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met
100 105 110
Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu
115 120 125
Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met
130 135 140
Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp
145 150 155 160
Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly
165 170 175
Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp
180 185 190
Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu
195 200 205
<210> 89
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 89
Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu
1 5 10 15
Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe
20 25 30
Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala
35 40 45
Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile
50 55 60
Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val
65 70 75 80
Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile
85 90 95
Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met
100 105 110
Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu
115 120 125
Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met
130 135 140
Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn
145 150 155 160
Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly
165 170 175
Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys
180 185 190
Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu
195 200 205
<210> 90
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 90
Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala
20 25 30
Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu
115 120 125
Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala
130 135 140
Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 91
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 91
Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala
20 25 30
Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu
115 120 125
Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala
130 135 140
Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 92
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<400> 92
Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala
20 25 30
Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu
115 120 125
Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala
130 135 140
Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 93
<211> 156
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<220>
<221> MISC_FEATURE
<222> (70)..(70)
<223> Xaa is A or K
<400> 93
Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp
1 5 10 15
Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile
20 25 30
Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala
35 40 45
Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu
50 55 60
Gly Met Pro Gly Lys Xaa Glu Lys Asp Lys Val Cys Ala His Glu Ala
65 70 75 80
Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile
85 90 95
Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys
100 105 110
Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr
115 120 125
Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu
130 135 140
Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu
145 150 155
<210> 94
<211> 209
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa is S or D
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is T or D
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is A or R
<220>
<221> MISC_FEATURE
<222> (85)..(85)
<223> Xaa is T or D
<220>
<221> MISC_FEATURE
<222> (119)..(119)
<223> Xaa is A or Q
<220>
<221> MISC_FEATURE
<222> (163)..(163)
<223> Xaa is S or D
<220>
<221> MISC_FEATURE
<222> (189)..(189)
<223> Xaa is T or R
<400> 94
Met Xaa Xaa Ile Asn Asn Gln Leu Lys Xaa Leu Lys Val Ile Pro Val
1 5 10 15
Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu
20 25 30
Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala
35 40 45
Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu
50 55 60
Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys
65 70 75 80
Glu Ala Gly Ala Xaa Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr
85 90 95
Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn
100 105 110
Asn Pro Ser Thr Val Glu Xaa Ala Leu Glu Met Gly Leu Thr Thr Leu
115 120 125
Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser
130 135 140
Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile
145 150 155 160
Thr Pro Xaa Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala
165 170 175
Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Xaa Asn Gly Glu
180 185 190
Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn
195 200 205
Pro
<210> 95
<211> 114
<212> PRT
<213> Artificial sequence
<220>
<223> Synthesis of
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> Xaa is T or D
<220>
<221> MISC_FEATURE
<222> (33)..(33)
<223> Xaa is K or E
<220>
<221> MISC_FEATURE
<222> (71)..(71)
<223> Xaa is S or D
<220>
<221> MISC_FEATURE
<222> (74)..(74)
<223> Xaa is R or E
<220>
<221> MISC_FEATURE
<222> (101)..(101)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (103)..(103)
<223> Xaa is N or D
<400> 95
Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Xaa Asp Val Pro
1 5 10 15
Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser
20 25 30
Xaa Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu
35 40 45
Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser
50 55 60
Ile Gly Gly Ile Glu Pro Xaa Lys Asn Xaa Asp His Ser Ala Val Leu
65 70 75 80
Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr
85 90 95
Ile His Phe Val Xaa Leu Xaa Gly Asp Asp Val Gly Trp Asn Gly Thr
100 105 110
Thr Phe
<210> 96
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa is Y or H
<220>
<221> MISC_FEATURE
<222> (82)..(82)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (87)..(87)
<223> Xaa is R or D
<220>
<221> MISC_FEATURE
<222> (105)..(105)
<223> Xaa is S or D
<220>
<221> MISC_FEATURE
<222> (119)..(119)
<223> Xaa is R or E
<220>
<221> MISC_FEATURE
<222> (121)..(121)
<223> Xaa is R or E
<220>
<221> MISC_FEATURE
<222> (124)..(124)
<223> Xaa is A or D
<220>
<221> MISC_FEATURE
<222> (126)..(126)
<223> Xaa is H or D
<220>
<221> MISC_FEATURE
<222> (128)..(128)
<223> Xaa is R or E
<220>
<221> MISC_FEATURE
<222> (150)..(150)
<223> Xaa is A or N
<400> 96
Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala
20 25 30
Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa
115 120 125
Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala
130 135 140
Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 97
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<220>
<221> MISC_FEATURE
<222> (126)..(126)
<223> Xaa is T or D
<220>
<221> MISC_FEATURE
<222> (139)..(139)
<223> Xaa is Q or E
<220>
<221> MISC_FEATURE
<222> (142)..(142)
<223> Xaa is K or E
<220>
<221> MISC_FEATURE
<222> (160)..(160)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (163)..(163)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (166)..(166)
<223> Xaa is E or K
<220>
<221> MISC_FEATURE
<222> (169)..(169)
<223> Xaa is D or K
<220>
<221> MISC_FEATURE
<222> (179)..(179)
<223> Xaa is S, D or K
<220>
<221> MISC_FEATURE
<222> (183)..(183)
<223> Xaa is K or E
<220>
<221> MISC_FEATURE
<222> (185)..(185)
<223> Xaa is T, D, or K
<220>
<221> MISC_FEATURE
<222> (192)..(192)
<223> Xaa is D or K
<220>
<221> MISC_FEATURE
<222> (195)..(195)
<223> Xaa is A, E, or K
<220>
<221> MISC_FEATURE
<222> (198)..(198)
<223> Xaa is E or K
<220>
<221> MISC_FEATURE
<222> (199)..(199)
<223> Xaa is E or K
<400> 97
Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu
1 5 10 15
Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe
20 25 30
Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala
35 40 45
Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile
50 55 60
Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val
65 70 75 80
Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile
85 90 95
Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met
100 105 110
Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu
115 120 125
Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met
130 135 140
Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa
145 150 155 160
Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly
165 170 175
Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa
180 185 190
Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu
195 200 205
<210> 98
<211> 157
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa is Y or H
<220>
<221> MISC_FEATURE
<222> (38)..(38)
<223> Xaa is A or R
<220>
<221> MISC_FEATURE
<222> (82)..(82)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (87)..(87)
<223> Xaa is R or D
<220>
<221> MISC_FEATURE
<222> (97)..(97)
<223> Xaa is N or D
<220>
<221> MISC_FEATURE
<222> (105)..(105)
<223> Xaa is S, N, or D
<220>
<221> MISC_FEATURE
<222> (119)..(119)
<223> Xaa is R, E, or N
<220>
<221> MISC_FEATURE
<222> (121)..(121)
<223> Xaa is R, E, or D
<220>
<221> MISC_FEATURE
<222> (122)..(122)
<223> Xaa is K or D
<220>
<221> MISC_FEATURE
<222> (124)..(124)
<223> Xaa is K or D
<220>
<221> MISC_FEATURE
<222> (126)..(126)
<223> Xaa is H or D
<400> 98
Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val
1 5 10 15
Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala
20 25 30
Phe Glu Ala Ala Met Xaa Asp Ile Gly Gly Asp Arg Phe Ala Val Asp
35 40 45
Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr
50 55 60
Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val
65 70 75 80
Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile
85 90 95
Xaa Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser
100 105 110
Ala Val Leu Thr Pro His Xaa Tyr Xaa Xaa Ser Xaa Ala Xaa Thr Leu
115 120 125
Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala
130 135 140
Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala
145 150 155
<210> 99
<211> 142
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 99
taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat 60
acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta 120
agtataagaa ggagatatac tt 142
<210> 100
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 100
taaagaagga gatatcat 18
<210> 101
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic
<400> 101
tgagaaggag atatcat 17
<210> 102
<211> 365
<212> PRT
<213> Intelligent people
<400> 102
Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe
1 5 10 15
Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr
20 25 30
His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp
35 40 45
Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu
50 55 60
Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val
65 70 75 80
Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys
85 90 95
Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr
100 105 110
Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val
115 120 125
Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp
130 135 140
Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile
145 150 155 160
Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr
165 170 175
Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg
180 185 190
Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys
195 200 205
Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe
210 215 220
Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu
225 230 235 240
Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser
245 250 255
Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His
260 265 270
Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val
275 280 285
Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val
290 295 300
Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu
305 310 315 320
Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg
325 330 335
Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp
340 345 350
Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala
355 360 365
<210> 103
<211> 62
<212> PRT
<213> Intelligent people
<400> 103
Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe
1 5 10 15
Leu Leu Pro Gly Ser Leu Gly Phe Ala Cys Lys Thr Ala Asn Gly Thr
20 25 30
Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala
35 40 45
Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser
50 55 60
<210> 104
<211> 574
<212> PRT
<213> human respiratory syncytial virus type A (A2 strain)
<400> 104
Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro
100 105 110
Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr
115 120 125
Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn
195 200 205
Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val
370 375 380
Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460
Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn
565 570
<210> 105
<211> 64
<212> PRT
<213> human respiratory syncytial virus type A (A2 strain)
<400> 105
Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Gly Phe Ala Cys Lys Thr Ala Asn
20 25 30
Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn
35 40 45
Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser
50 55 60
<210> 106
<211> 178
<212> PRT
<213> Intelligent people
<400> 106
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val
1 5 10 15
Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His
20 25 30
Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
35 40 45
Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu
50 55 60
Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys
65 70 75 80
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro
85 90 95
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu
100 105 110
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg
115 120 125
Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn
130 135 140
Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu
145 150 155 160
Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile
165 170 175
Arg Asn
<210> 107
<211> 57
<212> PRT
<213> Intelligent people
<400> 107
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val
1 5 10 15
Arg Ala Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly
20 25 30
Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val
35 40 45
Gly Gln Asn Leu Val Val Asp Leu Ser
50 55
<210> 108
<211> 562
<212> PRT
<213> influenza A virus (A strain/Japan/305/1957H 2N 2)
<400> 108
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp
1 5 10 15
Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp
20 25 30
Thr Asn Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu
35 40 45
Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro
50 55 60
Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro
65 70 75 80
Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu
85 90 95
Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp
100 105 110
Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys
115 120 125
Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly
130 135 140
Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn
145 150 155 160
Met Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly
165 170 175
Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val
180 185 190
His His Pro Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val
195 200 205
Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr
210 215 220
Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met
225 230 235 240
Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu
245 250 255
Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys
260 265 270
Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys
275 280 285
Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro
290 295 300
Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val
305 310 315 320
Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln
325 330 335
Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
340 345 350
Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn
355 360 365
Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala
370 375 380
Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn
385 390 395 400
Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn Leu Glu Arg Arg
405 410 415
Leu Glu Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val Trp
420 425 430
Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu
435 440 445
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met
450 455 460
Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe
465 470 475 480
Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr
485 490 495
Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu
500 505 510
Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala
515 520 525
Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala
530 535 540
Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
545 550 555 560
Cys Ile
<210> 109
<211> 54
<212> PRT
<213> influenza A virus (A strain/Japan/305/1957H 2N 2)
<400> 109
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Phe
1 5 10 15
Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser
20 25 30
Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn
35 40 45
Leu Val Val Asp Leu Ser
50

Claims (73)

1. A recombinant mammalian cell comprising a polynucleotide encoding a polypeptide derived from escherichia coli (e.
2. The recombinant cell of claim 1, wherein the polypeptide is derived from e.
3. The recombinant cell of claim 2, wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide.
4. The recombinant cell of claim 2, wherein the polypeptide comprises a phenylalanine residue within the first 20 residue positions of the N-terminus.
5. The recombinant cell of claim 2, wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide.
6. The recombinant cell of claim 5, wherein the polypeptide does not comprise a glycine residue immediately preceding the phenylalanine residue at position 1 of the polypeptide.
7. The recombinant cell of claim 2, wherein the polypeptide does not comprise an N-glycosylation site at position 7 of the polypeptide.
8. The recombinant cell of claim 6, wherein the polypeptide does not comprise an Asn residue at position 7 of the polypeptide.
9. The recombinant cell of claim 8, wherein the polypeptide comprises a residue at position 7 selected from the group consisting of Ser, Asp, Thr, and Gln.
10. The recombinant cell of claim 5, wherein the polypeptide does not comprise an N-glycosylation site at position 70 of the polypeptide.
11. The recombinant cell of claim 10, wherein the polypeptide does not comprise an Asn residue at position 70 of the polypeptide.
12. The recombinant cell of claim 10, wherein the polypeptide does not comprise a Ser residue at position 70 of the polypeptide.
13. The recombinant cell of claim 1, wherein the polypeptide comprises a residue substitution at an N-glycosylation site of the polypeptide selected from the group consisting of Ser, Asp, Thr, and gin.
14. The recombinant cell of claim 13, wherein the N-glycosylation site comprises position N235 of the polypeptide.
15. The recombinant cell of claim 13, wherein the N-glycosylation site comprises position N228 of the polypeptide.
16. The recombinant cell of claim 13, wherein the N-glycosylation site comprises position N235 and position N228 of the polypeptide.
17. The recombinant cell of claim 2, wherein the polypeptide comprises SEQ ID No. 3.
18. The recombinant cell of claim 2, wherein the polypeptide comprises SEQ ID No. 2.
19. The recombinant cell of claim 1, wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide.
20. The recombinant cell of claim 19, wherein said aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val.
21. The recombinant cell of claim 1, wherein the polypeptide comprises a fragment of FimH.
22. The recombinant cell of claim 21, wherein the polypeptide comprises the lectin domain of FimH.
23. The recombinant cell of claim 22, wherein the lectin domain comprises a mass of about 17022 daltons.
24. The recombinant cell of claim 1, wherein the polypeptide is complexed to a FimC polypeptide or fragment thereof.
25. The recombinant cell of claim 24, wherein the FimC polypeptide or fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or fragment thereof.
26. The recombinant cell of claim 2, wherein the polypeptide is in a low affinity conformation.
27. The recombinant cell of claim 2, wherein the polypeptide is stabilized by FimG.
28. The recombinant cell of claim 2, wherein the polypeptide is stabilized by the donor chain peptide (DsG) of FimG.
29. The recombinant cell of claim 28, wherein the polynucleotide sequence further encodes a linker sequence.
30. The recombinant cell of claim 29, wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues.
31. The recombinant cell of claim 29, wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues.
32. The recombinant cell of claim 29, wherein the linker comprises 7 amino acid residues.
33. The recombinant cell of claim 1, wherein the polypeptide does not comprise a signal peptide selected from the group consisting of: the native FimH leader peptide, influenza hemagglutinin signal peptide, and human respiratory syncytial virus a (strain a 2) fusion glycoprotein F0 signal peptide.
34. The recombinant cell of claim 1, wherein the polypeptide comprises a murine IgK signal peptide sequence.
35. The recombinant cell of claim 1, wherein the polypeptide comprises any signal peptide sequence selected from the group consisting of the signal peptide of the human IgG receptor FcRn large subunit p51 and the signal peptide of the human IL10 protein.
36. The recombinant cell of claim 2, wherein the polypeptide comprises an arginine to proline mutation at amino acid position 60 according to the numbering of SEQ ID NO 3 (R60P).
37. The recombinant cell of claim 1, wherein the expression level of the polypeptide is higher than the expression level of a corresponding wild-type polypeptide expressed in the periplasm of a wild-type E.
38. The recombinant cell of claim 1, wherein the polypeptide is expressed at a level greater than 10 mg/L.
39. The recombinant cell of claim 1, wherein the polynucleotide sequence is integrated into the genomic DNA of the mammalian cell.
40. The recombinant cell of claim 1, wherein the polynucleotide sequence is codon optimized for expression in the cell.
41. The recombinant cell of claim 1, wherein the cell is a human embryonic kidney cell.
42. The recombinant cell of claim 40, wherein the human embryonic kidney cell comprises a HEK293 cell.
43. The recombinant cell of claim 42, wherein the HEK293 cell is selected from any one of HEK293T, HEK293TS and HEK293E cells.
44. The recombinant cell of claim 1, wherein the cell is a CHO cell.
45. The recombinant cell of claim 44, wherein the CHO cell is a CHO-K1 cell, a CHO-DUXB11 cell, a CHO-DG44 cell, or a CHO-S cell.
46. The recombinant cell of claim 1, wherein the polypeptide is soluble.
47. The recombinant cell of claim 1, wherein the polypeptide is secreted from the cell.
48. The recombinant cell of claim 2, wherein the polypeptide comprises the N28Q substitution according to numbering of SEQ ID NO 1.
49. The recombinant cell of claim 2, wherein the polypeptide comprises the N28D substitution according to numbering of SEQ ID NO 1.
50. The recombinant cell of claim 2, wherein the polypeptide comprises the N28S substitution according to numbering of SEQ ID NO 1.
51. The recombinant cell of claim 2, wherein the polypeptide comprises a substitution selected from any one of N28Q, V48C, and L55C according to the numbering of SEQ ID NO 1.
52. The recombinant cell of claim 2, wherein the polypeptide comprises the substitution N92S according to the numbering of SEQ ID NO 1.
53. The recombinant cell of claim 1, wherein the FimH-derived polypeptide or fragment thereof comprises a substitution selected from any one of V48C and L55C, according to the numbering of SEQ ID No. 1.
54. A culture comprising the recombinant cell of claim 1, wherein the volume of the culture is at least 5 liters.
55. The culture of claim 49, wherein the polypeptide or fragment thereof is produced at a yield of at least 0.05 g/L.
56. The culture of claim 55, wherein the polypeptide or fragment thereof is produced at a yield of at least 0.10 g/L.
57. A method for producing a polypeptide derived from escherichia coli, or a fragment thereof, comprising culturing a recombinant mammalian cell according to claim 1 under suitable conditions, thereby expressing the polypeptide or fragment thereof; and harvesting the polypeptide or fragment thereof.
58. The method of claim 57, further comprising purifying the polypeptide or fragment thereof.
59. The method of claim 57, wherein the cell comprises a nucleic acid encoding any one of the following sequences: 5, 6, 7, 8 and 27.
60. The method of claim 57, wherein the yield of said polypeptide or fragment thereof is at least 0.05 g/L.
61. The method of claim 57, wherein the yield of said polypeptide or fragment thereof is at least 0.10 g/L.
62. A composition comprising a polypeptide having at least 70% identity to any one of the following sequences: 1, 2, 3, 4, 20, 23, 24, 26, 28 and 29.
63. The composition of claim 62, further comprising a saccharide comprising a structure selected from any one of the formulae in Table 1.
64. The composition of claim 63, wherein the saccharide is covalently bound to a carrier protein.
65. The composition of claim 64, wherein the carrier protein is selected from any one of: poly (L-lysine), CRM197Diphtheria Toxin Fragment B (DTFB), DTFB C8, Diphtheria Toxoid (DT), Tetanus Toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid or exotoxin a from Pseudomonas aeruginosa (Pseudomonas aeruginosa); detoxified exotoxin a (epa) of pseudomonas aeruginosa, Maltose Binding Protein (MBP), detoxified hemolysin a of staphylococcus aureus (s. aureus), aggregation factor a, aggregation factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae (Streptococcus pneumoniae) pneumolysin and detoxified variants thereof, campylobacter jejuni (c.jejuni) AcrA, and campylobacter jejuni native glycoproteins.
66. The composition of claim 64, wherein the carrier protein is CRM197。
67. The composition of claim 64, wherein the carrier protein is Tetanus Toxoid (TT).
68. The composition of claim 64, wherein the carrier protein is poly (L-lysine).
69. The composition of claim 64, wherein the saccharide is covalently bound to a carrier protein by a reductive amination reaction.
70. The composition of claim 64, wherein the saccharide is covalently bound to a carrier protein by CDAP chemistry.
71. The composition of claim 64, wherein the saccharide is conjugated to a covalent binding carrier protein through a single-ended linkage.
72. The composition of claim 64, wherein the saccharide is covalently bound to a carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer.
73. A polypeptide comprising an amino acid sequence selected from the group consisting of: 5, 6, 7, 8 and 27.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115671274A (en) * 2022-09-29 2023-02-03 普大生物科技(泰州)有限公司 Method for linking carrier protein and polysaccharide into conjugate by covalent bond and application

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2021223184A1 (en) * 2020-02-23 2022-08-18 Pfizer Inc. Escherichia coli compositions and methods thereof
CA3199610A1 (en) * 2020-10-27 2022-05-05 Pfizer Inc. Escherichia coli compositions and methods thereof
AU2021392894A1 (en) * 2020-12-02 2023-06-29 Glaxosmithkline Biologicals Sa Donor strand complemented fimh
US20220202923A1 (en) 2020-12-23 2022-06-30 Pfizer Inc. E. coli fimh mutants and uses thereof
WO2023111907A1 (en) 2021-12-17 2023-06-22 Pfizer Inc. Polynucleotide compositions and uses thereof
WO2023227608A1 (en) * 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004148A2 (en) * 1999-07-13 2001-01-18 Medimmune, Inc. Donor strand complemented pilin and adhesin broad-based vaccines
US20020150587A1 (en) * 2000-07-07 2002-10-17 Solomon Langermann FimH adhesin proteins and methods of use
US20030158141A1 (en) * 2001-11-26 2003-08-21 Matthias Gromeier Genetically stable expression vector
CN1758919A (en) * 2003-03-28 2006-04-12 金伯利-克拉克环球有限公司 Compositions comprising hyaluronic acid for the prevention of urogenital infections

Family Cites Families (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4709017A (en) 1985-06-07 1987-11-24 President And Fellows Of Harvard College Modified toxic vaccines
US4950740A (en) 1987-03-17 1990-08-21 Cetus Corporation Recombinant diphtheria vaccines
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
NZ230747A (en) 1988-09-30 1992-05-26 Bror Morein Immunomodulating matrix comprising a complex of at least one lipid and at least one saponin; certain glycosylated triterpenoid saponins derived from quillaja saponaria molina
DE3841091A1 (en) 1988-12-07 1990-06-13 Behringwerke Ag SYNTHETIC ANTIGENS, METHOD FOR THEIR PRODUCTION AND THEIR USE
ES2055785T3 (en) 1989-01-17 1994-09-01 Eniricerche Spa SYNTHETIC PEPTIDES AND THEIR USE AS UNIVERSAL VEHICLES FOR THE PREPARATION OF IMMUNOGENOUS CONJUGATES SUITABLE FOR THE DEVELOPMENT OF SYNTHETIC VACCINES.
CA2063271A1 (en) 1989-07-14 1991-01-15 Subramonia Pillai Cytokine and hormone carriers for conjugate vaccines
IT1237764B (en) 1989-11-10 1993-06-17 Eniricerche Spa SYNTHETIC PEPTIDES USEFUL AS UNIVERSAL CARRIERS FOR THE PREPARATION OF IMMUNOGENIC CONJUGATES AND THEIR USE FOR THE DEVELOPMENT OF SYNTHETIC VACCINES.
SE466259B (en) 1990-05-31 1992-01-20 Arne Forsgren PROTEIN D - AN IGD BINDING PROTEIN FROM HAEMOPHILUS INFLUENZAE, AND THE USE OF THIS FOR ANALYSIS, VACCINES AND PURPOSE
IL98715A0 (en) 1990-08-13 1992-07-15 American Cyanamid Co Filamentous hemaglutinin of bodetella pertussis as a carrier molecule for conjugate vaccines
IT1262896B (en) 1992-03-06 1996-07-22 CONJUGATE COMPOUNDS FORMED FROM HEAT SHOCK PROTEIN (HSP) AND OLIGO-POLY-SACCHARIDES, THEIR USE FOR THE PRODUCTION OF VACCINES.
JP3506431B2 (en) 1992-05-06 2004-03-15 プレジデント アンド フェローズ オブ ハーバード カレッジ Diphtheria toxin receptor binding domain
UA40597C2 (en) 1992-06-25 2001-08-15 Смітклайн Бічем Байолоджікалс С.А. Vaccine composition, method for treatment of mammals, diseased or receptive to the infection, method for treatment of mammals with cancer, method for production of vaccine composition, composition of adjuvants
IL102687A (en) 1992-07-30 1997-06-10 Yeda Res & Dev Conjugates of poorly immunogenic antigens and synthetic pepide carriers and vaccines comprising them
ES2231770T3 (en) 1993-03-05 2005-05-16 Wyeth Holdings Corporation NEW PLASMIDS FOR THE PRODUCTION OF CRM PROTEIN AND DIFTERIC TOXIN.
ATE204762T1 (en) 1993-03-23 2001-09-15 Smithkline Beecham Biolog VACCINE COMPOSITIONS CONTAINING 3-0-DEAZYLATED MONOPHOSPHORYL LIPID A
GB9326253D0 (en) 1993-12-23 1994-02-23 Smithkline Beecham Biolog Vaccines
US6455673B1 (en) 1994-06-08 2002-09-24 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
US5917017A (en) 1994-06-08 1999-06-29 President And Fellows Of Harvard College Diphtheria toxin vaccines bearing a mutated R domain
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
CA2560114A1 (en) 1994-07-15 1996-02-01 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
AUPM873294A0 (en) 1994-10-12 1994-11-03 Csl Limited Saponin preparations and use thereof in iscoms
GB9513261D0 (en) 1995-06-29 1995-09-06 Smithkline Beecham Biolog Vaccines
AUPO517897A0 (en) 1997-02-19 1997-04-11 Csl Limited Chelating immunostimulating complexes
WO1998037919A1 (en) 1997-02-28 1998-09-03 University Of Iowa Research Foundation USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE IN THE TREATMENT OF LPS-ASSOCIATED DISORDERS
CA2301575C (en) 1997-05-20 2003-12-23 Ottawa Civic Hospital Loeb Research Institute Vectors and methods for immunization or therapeutic protocols
GB9712347D0 (en) 1997-06-14 1997-08-13 Smithkline Beecham Biolog Vaccine
GB9713156D0 (en) 1997-06-20 1997-08-27 Microbiological Res Authority Vaccines
EP1009382B1 (en) 1997-09-05 2003-06-18 GlaxoSmithKline Biologicals S.A. Oil in water emulsions containing saponins
US6303114B1 (en) 1998-03-05 2001-10-16 The Medical College Of Ohio IL-12 enhancement of immune responses to T-independent antigens
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
HUP0101619A3 (en) 1998-04-09 2003-11-28 Smithkline Beecham Biolog Adjuvant compositions
GB9817052D0 (en) 1998-08-05 1998-09-30 Smithkline Beecham Biolog Vaccine
KR100629028B1 (en) 1998-10-16 2006-09-26 글락소스미스클라인 바이오로지칼즈 에스.에이. Adjuvant systems and vaccines
CA2355364C (en) 1998-12-21 2014-03-18 Medimmune, Inc. Streptococcus pneumoniae proteins and immunogenic fragments for vaccines
CZ303675B6 (en) 1998-12-23 2013-02-27 Id Biomedical Corporation Of Quebec Isolated polynucleotide, vector, host cell, production process, isolated polypeptide, chimeric polypeptide, vaccine composition and use
AUPP807399A0 (en) 1999-01-08 1999-02-04 Csl Limited Improved immunogenic lhrh composition and methods relating thereto
EP2204186B1 (en) 1999-02-17 2016-04-06 CSL Limited Immunogenic complexes and methods relating thereto
AR022963A1 (en) 1999-03-19 2002-09-04 Smithkline Beecham Biolog VACCINE
WO2000061761A2 (en) 1999-04-09 2000-10-19 Techlab, Inc. Recombinant clostridium toxin a protein carrier for polysaccharide conjugate vaccines
WO2000062800A2 (en) 1999-04-19 2000-10-26 Smithkline Beecham Biologicals Sa Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide
CA2383413A1 (en) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Use of combination of polyoxyethylene sorbitan ester and octoxynol as adjuvant and its use in vaccines
KR20020048942A (en) 1999-09-24 2002-06-24 장 스테판느 Adjuvant comprising a polyoxyethylene alkyl ether or ester and at least one nonionic surfactant
GB0007432D0 (en) 2000-03-27 2000-05-17 Microbiological Res Authority Proteins for use as carriers in conjugate vaccines
JP5051959B2 (en) 2000-06-20 2012-10-17 アイディー バイオメディカル コーポレイション オブ ケベック Streptococcus antigen
WO2002091998A2 (en) 2001-05-11 2002-11-21 Aventis Pasteur, Inc. Novel meningitis conjugate vaccine
EP1456231A2 (en) 2001-12-20 2004-09-15 Shire Biochem Inc. Streptococcus antigens
NZ541969A (en) 2003-03-13 2008-01-31 Glaxosmithkline Biolog Sa Purifying pneumolysin from Streptococcus pneumoniae in a single chromatographic step by binding it to a hydrophobic interaction column in the presence of detergent and high salt
EP1603950A2 (en) 2003-03-17 2005-12-14 Wyeth Holdings Corporation Mutant cholera holotoxin as an adjuvant and an antigen carrier protein
JP2008506683A (en) 2004-07-18 2008-03-06 コーリー ファーマシューティカル グループ， リミテッド Methods and compositions for inducing innate immune responses
KR100958505B1 (en) 2004-07-18 2010-05-17 씨에스엘 리미티드 Immuno stimulating complex and oligonucleotide formulations for inducing enhanced interferon-gamma responses
US7955605B2 (en) 2005-04-08 2011-06-07 Wyeth Llc Multivalent pneumococcal polysaccharide-protein conjugate composition
IL308456A (en) 2005-04-08 2024-01-01 Wyeth Llc Multivalent pneumococcal polysacharide-protein conjugate composition
US20070184072A1 (en) 2005-04-08 2007-08-09 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US7709001B2 (en) 2005-04-08 2010-05-04 Wyeth Llc Multivalent pneumococcal polysaccharide-protein conjugate composition
KR101579947B1 (en) 2007-06-26 2015-12-28 글락소스미스클라인 바이오로지칼즈 에스.에이. Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates
RU2536248C2 (en) 2009-04-30 2014-12-20 Коули Фармасьютикал Груп, Инк. Pneumococcal vaccine and using it
PT3421051T (en) 2012-08-16 2020-06-26 Pfizer Glycoconjugation processes and compositions
KR102630357B1 (en) 2017-02-17 2024-01-30 론자 리미티드 Multi-site SSI cells with difficult protein expression

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004148A2 (en) * 1999-07-13 2001-01-18 Medimmune, Inc. Donor strand complemented pilin and adhesin broad-based vaccines
US20020150587A1 (en) * 2000-07-07 2002-10-17 Solomon Langermann FimH adhesin proteins and methods of use
US20030158141A1 (en) * 2001-11-26 2003-08-21 Matthias Gromeier Genetically stable expression vector
CN1758919A (en) * 2003-03-28 2006-04-12 金伯利-克拉克环球有限公司 Compositions comprising hyaluronic acid for the prevention of urogenital infections

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王威;刘斌;刘雪倩;冯露;孙丹;王磊;: "大肠杆菌O85 O-抗原基因簇序列破译和特异分子标识的筛选", 中国人兽共患病学报, no. 01, pages 39 - 44 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115671274A (en) * 2022-09-29 2023-02-03 普大生物科技(泰州)有限公司 Method for linking carrier protein and polysaccharide into conjugate by covalent bond and application
CN115671274B (en) * 2022-09-29 2024-03-12 普大生物科技(泰州)有限公司 Method for covalent bond connection of carrier protein and polysaccharide into conjugate and application

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